Leukemia cell lines: In vitro models for the study of chronic myeloid leukemia

~ ) Pergamon

Leukemia Research Vol. 18, No. 12, pp. 919-927, 1994

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00

0145-2126(94)00107-3 COMMENTARY

LEUKEMIACELLL I N E S :

IN VITRO MODELS FOR THE STUDY OF

CHRONIC MYELOID LEUKEMIA Hans G. Drexler German Collection of Micro-organisms & Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany

(Received 19 April 1994. Accepted 19 May 1994)

Chronic myeloid leukemia (CML) serves as a valuable paradigm for studying the pathogenesis, evolution and treatment of human malignancy. This disease has stood as a model for understanding the origins of cancer, first at the chromosomal and recently at the molecular genetic level. CML is arguably the most carefully studied and best understood cancer in humans. CML not only served as a prototype neoplasm for basic research, but also for clinical studies designed to develop curative cancer treatment. Well established features of CML are an increased production of multiple types of mature blood cells and an origin in a pluripotent stem cell with the socalled Philadelphia (Ph) chromosome. The demonstration of the Ph chromosome in all blood cell types including B- and more recently T-lymphocytes provided unequivocal evidence that the neoplastic clone originates in a very primitive pluripotent hematopoietic cell. The Ph chromosome (that is the reciprocal translocation t(9;22) (q34;q11)) is found with great specificity in CML cells. The molecular consequences of the Ph translocation are now well established: two genes, normally each located on a separate chromosome (the BCR gene on chromosome 22 and the ABL gene on chromosome 9), become fused to form a new chimeric BCR/ABL gene that is expressed as a p210 fusion protein with increased tyrosine kinase activity. Most patients eventually enter the blastic transformation stage. The CML cells in this blast crisis exhibit a variety of characteristics including those

specifically detected in myeloid, lymphoid, erythroid, monocytic and megakaryocytic lineage cells attesting further to the multilineage involvement of CML cells.

CML cell lines Human leukemia cell lines have been used for 30 years as important research tools for the study of neoplastic transformation and hematopoietic cell differentiation [1-3]. Established leukemia cell lines with unique phenotypic or karyotypic features have been extremely useful models for the investigation of the molecular and biological characteristics of specific types of leukemia. Progress in the understanding of the molecular events associated with the Ph translocation has been facilitated by the ready availability of cells from permanent cell lines such as CML-derived cultures [4]. For example, the K-562 cell line that carries the t(9;22) was used to clone the ABL oncogene [5]. Another example is the cell line NALM-I: using this CML-derived cell line it could be shown definitively that CML blast crisis cells can have features associated with B-cell lineage (in fact, phenotypes of pre-B-cells), thus demonstrating the pluripotential of the CML cells [6, 7]. While the K-562 cell line was the first CML-derived continuous cell line, the recent establishment of a number of CML cell lines has significantly broadened the range of morphological, immunophenotypical, cytogenetic, molecular genetic, functional and other biological attributes. A search of my leukemia cell line database showed some three dozen entries. Table 1 lists these cell lines in alphabetical order according to the name of the cell line. Further, but less well described CML-derived cell lines are MC3, TS9;22 and YS9;22 with myeloid features and

Correspondence to: Dr Hans G. Drexler, DSM-German Collection of Micro-organisms and Cell Cultures, Mascheroder Weg 1B, D-38124 Braunschweig, Germany (Tel: +49 531 2616 160; Fax: +49 531 2616 150). 919

66 F 50F 62 M 37 M

53 F

67 F 40 M

GDM-I GM/SO JK-1 JOSK-M

K-562

KBM-5 KBM-7

PB BM

PE

PB BM tumor PB

BM

PB PB BM

Source*

BM PB BM PB PB

41 M 3F 44 F n.i. 62 F

57F

35 M 77 M

MOLM-1 NALM-1 RM10 RWLeu-4 T-33

Y-1K

YNd YOS-M

CML in erythroid BC CML in myeloid BC

CML in mcgakaryocytic blast crisis (?ME in BC CML in lymphoid BC CML in BC CML in BC CML in megakaryocytic BC CML in erythroid BC Erythroid megakaryocytic Erythroid Myeloid

Myeloid eosinophilic Erythroid megakaryocytic Megakaryocytic Pre-B-lymphoid Erythroid Monocytic Megakaryocytic

Myeloid Erythroidmegakaryocytic Erythroid eosinophilic

Myeloid Basophilic-erythroid

CML in BC CML in BC CML in myeloid BC CML in myeloidmegakaryocytic BC

Myeloid Erythroid

Monocytic Myeloid

Erythroid

Myeloid Myeloid Erythroid Monocytic

Myeloid

Pre-B-lymphoid T-lymphoid Myeloid

Lineage characteristics+

CML in monocytic BC CML in myeloid BC (after BMT) CML in BC CML in erythroid BC

CML in BC CML in lymphoid BC CML in BC (relapse after BMT) CML in BC (relapse after BMT) CML in myeloid BC CML in myeloid BC CML in erythroid BC CML in myelomonocytic BC CML in BC

Original disease*

Ph + Ph +

Ph+

Ph -~ Ph+ Ph+ Ph+: macrophage differentiation potential Ph+

Ph+ Ph+ Slightly different subclones Ph+: megakaryocytic diffcrcntiation potcntial Ph+: basophilic, erythroid, macrophagc differentiation potential Maerophage differentiation potential Ph + Ph+: erythroid, megakaryocytic, monocvtic differentiation potential Erythroid-megakaryocytic differcntiation potential Monoeytic dift;erentiation potential Ph+

Ph+, - , BCR/ABL+: myeloid, ervthroid. megakaryocytic differentiation potential Pht Ph+

Ph+: same patient as EM-2. but established from a different sample PhPh+: growth dependent on GM-CSF Ph+ P h-

Ph+ Ph , BCR/ABL+ Ph+

Ph ade ph a chro n )s) nc; Othcr rcmarks

33 34

33

28 6, 7 29 30.31 32

26 27

26

23 24 25

21 22

19 20

16. 17 18

15

1l 12 13 14

l(I

8 9 10

Refercncc

Abbreviations: BC. blast crisis; BM, bone marrow; BMT, bone marrow transplantation: CML, chronic myeloid leukemia; GM-CSF, granulocyte-macrophage colony stimulating factor: n.i., not indicated: PB, peripheral blood; PE, pleural effusion. * N a m e o f cel l l i n e , a g e a n d s e x o f p a t i e n t , s o u r c e of m a t e r i a l a n d d i s e a s e ( s u b ) t y p e o f t h e p a t i e n t as i n d i c a t e d in t h e original publication quoted. f L i n e a g e c h a r a c t e r i s t i c s o f t h e e s t a b l i s h e d c e l l l i n e as i n d i c a t e d b y m u l t i - p a r a m e t e r a n a l y s i s ( f o r e x a m p l e , m o r p h o l o g y , c y t o c h e m i s t r y , i m m u n o p h e n o t y p i n g , g e n o t y p i n g , m o l e c u l a r b i o l o g i c a l a n a l y s i s , f u n c t i o n a l a s s a y s , e t c . ) a n d d e s c r i b e d in t h e o r i g i n a l p u b l i c a t i o n o r in s u b s e q u e n t p u b l i c a t i o n s n o t q u o t e d h e r e . :~ P h = P h i l a d e l p h i a c h r o m o s o m e ( t r a n s l o c a t i o n 9 ; 2 2 ) ; B C R / A B L = f u s i o n g e n e r e s u l t i n g f r o m a 9 :2 2 t r a n s l o c a t i o n .

PB PB

PB

BM

PB PB

LAMA-88 (subclone) MEG-01 55 M

LAMA-87 (subclone)

KU-812-F (subclone) KYO- 1 22 M LAMA-84 29 F

KCL-22 32 F PE KH88 70 M PB B4D6, C2F8 (subclones) KOPM-28 64 F PB KU-812 38 M PB

5F

45 M 36F 5F

BV173 CML-TI EM-2

EM-3

Patient*

Cell line*

T a b l e 1. C M L - d e r i v e d c e l l l i n e s

x

Fig. 1. Morphological variability of CML-derived cell lines. (a) Cell line EM-2 with myeloid Features; (b) cell line K-562 with erythroid/muttilineage features; (c) cell line BV173 with 9re-B-cell lymphoid characteristics; (d) cell line CML-T1 with T-cell lymphoid characteristics (see also Table 1) (May-Gr/inwald-Giemsa staining, original magnification 150×).

CMk-dcrived cell lines

923

Table 2. Ph+ ALL-derived cell lines Cell line*

Patient* Source ~

ALL-1 ALL/MIK KOPM30 KOPN30bi LEF1 MR-87 NALM-20

6F 74F n.i. 8M 37 F 4M 62 M

BM PB PB BM PB BM PB

Original disease*

Lineage characteristics+

Ph Chromosomes

Pre-B-ALL Pre-B-ALL n.i. Common ALL ALL (L1) AML Pre-B-ALL

Pre-B-lymphoid Pre-B-lymphoid Myeloid Pre-B-lymphoid Pre-B-lymphoid Myeloid Pre-B-lymphoid

Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+

Subclones

m-bcr+ m-bcr+ m-bcr+ m-bcr+

NALM-24 OM9:22 PALL-1 PALI,-2 SUP-B13

42 F 16 F 24 M 45 M 9M

PB BM PB PB BM

Pre-B-ALL ALL Pre-B-ALL Pre-B-ALL Pre-B-ALL

Pre-B-lymphoid Pre-B-lymphoid Pre-B-lymphoid Pre-B-lymphoid Pre-B-lymphoid

Ph+ Ph+ Ph+ Ph+ Ph+

TOM-1 Z-119 Z-181

54 M 25 M 32 M

BM BM BM

Pre-B-AH, ALL ALL

Pre-B-tymphoid Ph+ m-bet÷ Pre-B-lymphoid Ph+ m-bcr+ Pre-B-lymphoid Ph+ m-bcr÷

NALM-21/'-22/-23 established at relapse NALM-25

SUP-B15 established at relapse

Reference 44 45 46 47 48 49 50 51 52 53, 54 54 55 56 57 57

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acutc myeloid leukemia: BM, bone marrow: n.i.. not indicated; PB, peripheral blood. * Name of cell line, age and sex of patient, source of material and disease (sub)type of the patient as indicated in the original publication quoted. ~ Lineage characteristics of the established cell line as indicated by multi-parameter analysis (e.g., morphology, cytochemistry, immunophenotyping, genotyping, molecular biological analysis, functional assays, etc.) and described in the original publication or in subsequent publications not quoted here. $ Ph, Philadelphia chromosome (translocation 9;22): m-bet, breakpoint on chromosome 22 in the minor breakpoint cluster region (m-bcr) leading to the p190 BCR/ABL fusion genc. H I M e g , Meg-J and SKH1 with megakaryocytic characteristics [35-38]. Two cell lines derived from patients with chronic myelomonocytic leukemia (CMML) were reported. The line T M M was established from the peripheral blood of a 62-year-old male with C M M L in blast crisis after myelodysplastic syndrome (MDS) and was described as expressing myeloid-associated parameters and as lacking both the Ph chromosome and a B C R / A B L rearrangement [39]. Possibly, the original neoplastic cells were overgrown by EpsteinBarr virus ( E B V ) transformed (normal) B-lymphocytes [40]. The second CMML-derived cell line P 3 9 / T S U G A N E stems from the peripheral blood of a 69-year-old man with C M M L that developed into an A M L F A B M2 [41]. These latter cells do not carry the t(9;22); molecular genetic studies were not reported. All the C M L cell lines reported (Table 1) have extensive structural and numerical chromosome abnormalities in addition to the presence of the Ph c h r o m o s o m e . Several cell lines have multiple copies of Ph (such as EM-2, EM-3). These cell lines carry often further chromosomal aberrations associated with C M L progression, for example an isochrom o s o m e of the long arm of chl7, i(17q), (such as

EM-2, KU-812) or trisomy 8 (such as KCL-22, KBM7). The occurrence and structure of the chimeric B C R / A B L fusion gene has been analyzed in many of the CML cell lines providing, as already pointed out, valuable material for these molecular investigations. Inactivation of the t u m o r suppressor gene p53 has been found in association with blastic transformation of CML. Interestingly, most myeloid C M L cell lines had inactivation of p53 t u m o r suppressor function (resulting from c h r o m o s o m e 17 deletion, i[17q], or point mutations) and most also produced mutant p53 proteins [42, 43]. Cell line model systems may prove useful in identifying additional novel mutations in C M L disease progression which would be difficult or impossible to obtain from fresh cells. However, it should be kept in mind that the c o m m o n approach for establishing continuous leukemia cell lines may select for skewed populations. Leukemia cell lines also provide insight into the regulation and modulation of normal hematopoiesis. But hematopoietic cell lines cannot be assumed to have completely normal mechanisms of differentiation and indeed are unlikely to do so. One striking feature that can be gathered from Table 1 is that different lineage characteristics are

924

H.G. Drexler

expressed by these CML cell lines or that the expression of these varied characteristics can be induced by certain biomodulators. Already at the morphological level there is a great variability between the CML cell lines (Figure 1). Among the various cell lines the cells giving rise to the continuous cell culture appear to be arrested at different stages of differentiation: for instance cell lines K-562 and LAMA-84 might be regarded as multipotential cells expressing granulomonocytic, erythroid and megakaryocytic markers constitutively or upon treatment with appropriate factors [26]; on the other hand, Y-1K cells are suggested to represent an early bipotential progenitor stage of the erythroid-megakaryocytic lineage displaying markers of both lineages which indicates that erythroid and megakaryocytic lineages are probably closely associated during early hematopoietic differentiation [33]. Ph-positive ALL cell lines In the context of the t(9;22) in CML, Ph-positive (Ph+) acute lymphoblastic leukemia (ALL) must be mentioned as well. In Ph+ ALL, the breakpoints on chromosome 22qll are variable with half of the cases displaying breakpoints within the so-called major breakpoint cluster region (M-bcr) similar to those seen in CML (and encoding the p210 BCR/ABL fusion protein) while the remaining samples show mainly rearrangements in the minor breakpoint cluster region (m-bcr) and translation of a p190 BCR/ ABL protein. A number of cell lines derived from patients with Ph+ ALL have been described and are listed in Table 2. It is obvious that all CML-derived cell lines (Table 1) have arisen from patients in blast crisis. Thus, to some extent, these lines are 'acute leukemia cell lines'. While this will not diminish their importance as models of CML, these cultures are, nevertheless, unlikely to reflect the pathophysiology of CML in chronic phase. It might be worthwhile to explore any salient differences between the CML and the Ph+ ALL cell lines, should there be any apart from the different bcr-breakpoints. Moreover, AML- and other ALL-derived cell lines may be probed in comparison with CML and Ph+ ALL cultures in order to, for instance, define exactly which structural or numerical cytogenetic alterations contribute to a given phenotypic expression of the cells independent or dependent of their origin from specific disease subtypes or stages. Summary The clinical importance of CML lies in its poor

responsiveness to chemotherapy which has proved highly effective in treating ALL. The scientific importance of CML resides in its role as a cancer prototype, permitting the identification of genes centrally involved in both neoplastic change and normal cellular differentiation. One of these genes, the fusion gene BCR/ABL resulting from the balanced translocation (9;22) has received wide attention owing to its intimate involvement in CML. Although a tremendous amount of data have been recently discovered about BCR/ABL, its exact role in leukemogenesis and normal hematopoiesis remains obscure. The study of CML cell lines has already been of considerable help in understanding the molecular events associated with the Ph chromosome [4]. Further advances are likely to be forthcoming, particularly at the molecular genetic, but also at the protein level. CML cell lines may offer an excellent means of addressing many issues as continuous cell lines represent an inexhaustible source of identical cell material that, in addition, can be made available to other researchers around the world. This overview on the thus far reported CMLderived cell lines supports the hypothesis that in some specimens of CML the target cells in which Ph translocation arises are not necessarily lineagerestricted committed progenitor cells, but are in fact in some (or all?) cases precommitted bipotential or multipotential progenitor or stem cells retaining the potential for differentiation in diverse hematopoietic directions [26]. In conclusion, established tumor cell lines with their unique phenotypic and karyotypic features have been extremely useful models for investigation of the molecular and biological characteristics of CML. Considerable progress in understanding the molecular and cell biology of CML has been achieved. Further advances in the knowledge of CML are expected to accrue with the productive use of these powerful research tools for many important unresolved issues. By so doing, these discoveries might open new avenues that promise to move clinicians closer to the goal of the prevention or cure of CML in all patients.

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