Int. J. Cancer: 19, 337-344 (1977)

TUMORIGENICITY OF HUMAN HEMATOPOIETIC CELL LINES IN ATHYMIC NUDE MICE Kenneth NILSSON l, Beppino C. GIOVANELLA *, John S. STEHLIN and George KLEIN The Wallenberg Laboratory, University of Uppsala, Box 562, S-7.51 22 Uppsala, Sweden; Cancer Research Laboratory, St. Joseph Hospital, Houston, Texas 77002, USA; and Institute of Tumor Biology, Karolinska Institutet, S-104 01 Stockholm, Sweden

Human hematopoietic cell lines, which had been classifiedon the basis of studies on clonality,and morphological, chromosomal and functional parameters as lymphoblastoid cell liner (LCL) of presumed nonneoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation t o nude mice and for capacity t o grow in semi-solid medium in vitro Recently established LCL failed t o grow both in nude mice and in agarose. I n contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity t o form colonies i n agarosc. Most lymphoma lines formed colonies i n agarote and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed t o grow. Both myeloma lines, however, formed colonies i n agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed t o grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown t o form tumors when inoculated subcutaneously in nude mice and had the capacity t o grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic i n nude mice. The capacity t o form colonies i n agarose i s not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur i n the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential t o grow subcutaneously in nude mice and in agarose in vitro. ( 3 ) The inconsistency with regard t o the capacity of some of the neoplastic cell lines t o grow i n nude mice o r i n agarose seems t o underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.

The vast majority of human hematopoietic cell lines carry Epstein-Barr virus (EBV) genomes as judged by nucleic acid hybridization (Nanoyama and Pagano, 1971 ; zur Hausen et al., 1972; Minowada et a[., 1974) and immunofluorescence staining for EBV nuclear antigen (EBNA) (Reedman and Klein, 1973). EBV-carrying lines have been derived from both neoplastic and non-neoplastic tissues. Recent studies on clonality (Fialkow et al., 1970; BCchet et al., 1974), morphology and functional

characteristics (Nilsson and PontBn, 1975) and chromosomes (Jarvis et al., 1974; Zech et al., 1976) suggest that two biologically different types of EBV genome-positive lymphoid lines exist. One, here termed “ lymphoblastoid ” cell line (LCL), is normal diploid and polyclonal and can be derived not only from normal donors but also from contaminating B-lymphoid cells in various tumours (Nilsson et al., 1970; Nilsson and PontBn, 1975). The other type of line, here designated Burkitt lymphoma (BL) line, is usually derived from African BL (Epstein, 1965; Nadkarni et al., 1969) or occasionally from BL occurring outside Africa (Gravel1 et al., 1976). The BL lines are considered to be of “ true ” neoplastic origin. The rare EBV genome-negative lines, derived from leukemias (Foley et al., 1965; Minowada et al., 1972; Lozzio and Lozzio, 1975), myelomas (Matsuoka et al., 1967; Nilsson et al., 1970), histiocytic (Epstein and Kaplan, 1974; Sundstrom and Nilsson, 1976), lymphocytic (Nilsson and Sundstrom, 1974) and exceptional EBV-negative Burkitt’s lymphoma (Klein et al., 1974) have all been representative of the tumor-cell population in vivo. The occurrence of karyotypic alteration in lymphoid cell lines during long-term culture, which may be followed by changes in phenotypic characteristics (Nilsson and PontCn, 1975) is a particular problem in attempting to make a distinction between the LCL and BL types of EBVpositive cell lines. The fact that EBV-carrying lymphoid lines could be derived both from Burkitt lymphomas and from apparently healthy donors has led to the assumption that the two are equivalent with regard to their malignant potential and the difference between the normal individual and the BL tumor patient is essentially determined by the host immune mechanism (for a review see Klein, 1975). Since the ultimate test for malignancy-inoculation into an autologous host-cannot be used, auxiliary tests were used to judge the possible neoplastic state of LCL and BL lines, including tests for the capacity to form colonies in agar in vitro (Imamura and Moore, 1968; Huang et al., 1969; Nilsson and PontCn, 1975) and heteroReceived: October 4, 1976.



transplantation in immunosuppressed or newborn hamsters, rats o r mice (Southam et al., 1969a, b; Huang et al., 1969; Levin et al., 1969; Christofinis, 1969; Imamura et al., 1970; Adams et al., 1970, 1971 and 1973; Deal et al., 1971). The picture that emerged from these studies was not very clear. Although lines of non-neoplastic origin usually grew poorly in agar while BL lines multiplied readily, exceptions were frequently encountered in both groups of lines. A similar inconsistency was found in the transplantation studies. However, these early studies did not take into full account the possibility of secondary chromosomal alterations in the cell lines. Neither was the existence of two biologically different types of EBV-carrying lines recognized.

The availability of a panel of well-characterized hematopoietic cell lines prompted us t o retest two parameters which are usually regarded as fairly reliable tests for malignancy: tumorigenicity after heterotransplantation t o nude mice (Pantelouris, 1968; Rygaard, 1969; Rygaard a n d Povlsen, 1969; Povlsen and Rygaard, 1971 ; Giovanella et al., 1972, 1974; Giovanella and Stehlin, 1974) and capacity for growth in semi-solid medium (Macpherson and Montagnier, 1964), The purpose of this investigation was t o study whether there was any difference between the two types of EBV-carrying lymphoid lines and the EBV-negative lines (the latter invariably of neoplastic origin) with regard t o tumorigenicity in nude mice a n d capacity for colony formation in agarose.


Cell line

Type of line


U-303 L

LCL, new line

U-43909 U-43912 U-43915 L7

LCL, new line LCL, new line LCL, new line LCL, new line

U-927 P1

LCL, new line

U-919 P1

LCL, new line


LCL, new line


LCL, new line

U-1157 u-1158 U-61 M U-255 Bm U-296 L Maku BJAB U-c262 U-47703 U-52002 U-984 Ramos EHR-A Ramos

LCL, new line LCL, new line LCL, old line LCL, old line LCL, old line LCL, old line BL, new line BL, new line BL, new line BL, new line BL, new line BL, American, new BL, American, new

EHR-B Ramos BL, American, new

I1 Wa Ramos

BL, American, new

Daudi Raji

BL, old line BL, old line


BL, old line

,ztLe + + ++ + + + + + + ++ ++ + ++ + + + -

+ +

++ +


Nilsson (1971~) Bkhet et al. (1974) Bkhet et al. (1974) Bdchet et al(1974) Zech, unpubl. Zech et al. (1976) Nilsson and Ponten, (1975) Glimelius ef al. (1975) Glimelius et al. (1 975) Nilsson, unpubl. Nilsson, unpubl. Ponten (1967) Nilsson (1971a,b) Nilsson (1971~) Yata and Klein, (1969) Klein et al. (1974) Nilsson and Pontdn (1975) Nilsson and Pontdn (1975) Nilsson and Pontdn (1975) Nilsson and P o n t h (1975) Klein et al. (1975) Klein et al. (1975) Klein et al. (1975) Klein et al. (1975) Klein et al. (1968) Pulvertaft (1965) Klein et al. (1972)


Normal diploid/Nilsson, unpubl. NT NT NT Normal diploid/Zech et al. (1976) Normal diploid/Zech et al (1976) Normal diploid/Zech, pers. comm. Normal diploid/Zech et al. (1976) Normai diploid/Zech et at. ( 1976) NT NT Aneuploid/Ponten (1967) Aneuploid/Nilsson, unpubl. Aneuploid/Zech et al. (1976) Aneuploid/Zech et al. (1976) Aneuploid/Zech et al. (1976) NT NT NT NT Aneuploid/Zech et al. (1976) . . Aneuploid/Klein et al. (1976) AneuDloidlKlein et al. (19%) ' Aneupoid/Klein et al (19%) Aneuploid/Zech et al. (1976) Aneuploid/Jarvis et al. (1974) Aneuploid/Toshima et al. (1967)

Colony Tumo- formarigeni- tion ,in city solid agarose


NT -


t5 -



++ + +



+ +



Colony Cell line

Type of line



Reference agarose

++ + ++

Rae1 Solubo

BL, old line BL, old line

Klein et al. (1972) Klein et al. (1972)

Namalva 1V

BL, old line

Reedman and Klein (1973)

Akuba Seraphina Odour EB 3

BL, old line BL, old line BL, old line BL, old line

Klein et al. (1972) Klein et al. (1975) Klein, unpubl. Epstein (1965)

NT Aneuploid/Manolov, pers, comm. Aneuploid/Manolov, pers. comrn. NT NT NT Aneuploid/Jarvis et al.

U-698 M

LL, old line

Nilsson and Sundstrom

Aneuploid/Zech et al. (1976)


Aneuploid/Zech et al. (1976)

+ +

Aneuploid/Ben-Bassat et al.


(1974) U-715 M

LL, old line

Nilsson and Sundstrom (1974)


LL, BL-like, old line HD, old line HL

zur Hausen (1972) Sundstrom and Nilsson


Myeloma, old line

Matsuoka et al. (1967)




Ben-Bassat et al. (1977)




Aneuploid/Zech et al. (1976) f 6 Aneuploid/Nilsson, unpubl. NT


Aneuploid/Huang et al.




Myeloma, old line ALL, old line

Nilsson et al. (1970) Minowada et a/.(1972)


ALL, old line

Schwenk and Schneider

U-266 B1


ALL, old line CML, old line

(1975) Foley et al. (1965)

Lozzio and Lozzio (1975)

Aneuoloid/Nilsson (1971b) AneuploidjHuang et al, (1974) NT '


+ -

Aneuploid/Foley et al. (1965) Aneuploid/Lozzioand Lozzio (1975)



+ + + + ++

Lines derived from the same donor -a LCL=lymphoblastoid cell line; BL =African Burkitt's lymphoma; LL =lymphocytic lymphoma; HD-Hodgkin's disease; HL-histiocytic lymphoma; ALL=acute lymphoblastic leukemia: CML=chronic myeloid leukemia.--" =colony formation of more than 0.1 % of the cells.-' N T = not tested.--l Tumor regressed.-O Avascular nodules.



Cell lines

EBV genome-carrying and EBV genome-negative human hematopoietic cell lines were studied. The former category included both lymphoblastoid (LCL) and African Burkitt's lymphoma (BL) cell lines (according to the classification proposed by Nilsson and PontBn, 1975). The LCL lines were all polyclonal (BCchet et al., 1974), normal diploid (Zech et al., 1976) and displayed functional, morphological and other phenotypic characteristics, which clearly distinguish them from the BL lines (Nilsson and PontBn, 1975; Nilsson et al., 1974; Glimelius et al., 1975; Fagraeus et al., 1975; Huber et al., 1976). The BL lines are monoclonal (Fialkow et al., 1970; BBchet et al., 1974), aneuploid (Zech et al., 1976) and are representative for the neoplastic prototype of the explanted uniclonal tumor. Since it is well established that long cultivation of LCL will result in secondary chromosal alterations (Zech et al., 1976) and changes of other phenotypic

characteristics (Nilsson and PontBn, 1975; Nilsson et al., 1974; Glimelius et al., 1975) both recently established (lo%). Among the group of BL lines, two old lines were tested and both formed large clusters of cells at a high cloning efficiency (>lo%). Of the five new BL lines tested only two formed colonies at a frequency of 2-5 %. Among the non-BL group of malignant lines all tested lines except U-698, U-715, Sg5 and U-937 formed colonies at a varying, but mostly high (>10%) cloning efficiency.



Heterotransplantation (Tables I and 11) All nine recently established LCL tested failed to grow. However, two out of four aneuploid LCL were tumorigenic. A similar, but less pronounced difference was found among the group of African BL lines. Of the new lines, three out of five were transplantable while eight out of 10 old lines showed successful growth. The EBV-negative American BL line Ramos and its EBV-converted sublines all grew equally well in the mice. In the group of EBV-negative non-BL tumor lines, six out of nine tested formed tumors with notable exceptions for the lymphoma line DG-75, the myeloma line U-266 B1 and the leukemia lines CCRF-CEM and JM. The great majority of the tumors appeared 10-30 days after inoculation and grew very rapidly afterwards to very large sizes, up to 20-40g. The only exceptions were RPMI 8226, U-296 L and S 95.


Cell type

LCL,new lines LCL,old lines BL,new lines BL, old lines LL, old lines ALL, old lines Myeloma, old lines HL,old line HD,old line CML,old line



Colony formation ~ in agarose


Karyotype ~


Normal diploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid Aneuploid


’ LCL=lymphoblastoid cell line (Nilsson and Pontkn, 1975); BL=Burkitt’s lymphoma; LL-lyrnphocytic lymphoma; ALL -acute lymphoblastic leukemia; HL =histiocytic lymphoma; HD = Hodgkin’s disease; CML =chronic myeloid leukemia.



Correlation between heterotransplantability and colony formation in agarose (Tables I and 11) Except for the diploid LCL no clear correlation between the two parameters was found. However, it is notable that the two secondarily altered lines U-255 Bm and U-296 L grew both in the nude mice and in vitro while the similarly aneuploid U-61 M failed t o proliferate in both test systems. Among the malignant lines no clear correlation between tumorigenicity and capacity for growth in agar was found. DISCUSSION

A clear difference in tumorigenic potential between the various types of EBV-carrying lymphoid cell lines was noted. N o diploid LCL formed a tumor while a majority of the aneuploid LCL and the lines of neoplastic origin (BL lines) did. The BL lines behaved in this respect like the EBV-negative lymphoma, leukemia and myeloma lines. Earlier findings that LCL from normal donors can be tumorigenic in animals (Christofinis, 1969; A d a m et al., 1970; Imamura et al., 1970; Adams et al., 1973) may be due to the use of chromasomally altered lines in the tests. Alternative explanations may be that different animal species and inoculation routes were used. That the route of inoculation is indeed of importance for successful heterotransplantation of human lymphoma cells has been shown by Arnstein et al. (1974) and Epstein et al. (1976). In these studies lymphoma cells which failed to grow subcutaneously produced tumors when inoculated intracerebrally. Whether our short-term cultured LC1 will behave similarly when inoculated intracerebrally is presently being tested. The lack of tumorigenicity for diploid LCL, together with the absence of growth in agarose, confirm the assumption that diploid LCL, although immortal in tissue culture, d o not behave as autonomous tumor cells in vivo (Nilsson and PontBn, 1975). This also been suggested by Belpomme et al. (1972) and Moore and Gerner (1970), who inoculated up to 300 g of LCL cells into the autologous donors and failed t o observe any progressive growth in vivo. It is, however, justified to question the validity of the capacity for tumor formation at least subcutaneously in the nude mouse as a criterion for malignancy especially since some (eight out of 25) of the lines of proven malignant ancestry failed to grow. Also, it is possible that the diploid LCL may grow at a different inoculation site and/or may be more sensitive to non-T-cell mediated immune lysis. There is no clear correlation between capacity for growth in agarose and tumorigenicity in nude mice for the neoplastic hematopoietic cell lines although this was true for the diploid LCL. Similar findings with other types of malignant human cells


have been reported (Stiles et al., 1976), while Freedman and Shin (1974) found a good correlation between growth in agar and in the nude mouse for human and mouse carcinoma cells. Taken together these facts suggest that capacity for tumor formation in nude mice must always be considered as one of several malignancy-related properties. In this view, " takes " would only occur with malignant cells, whereas the absence of growth may or may not indicate the lack of a malignant potential. The finding that the aneuploid LCL were often tumorigenic and have the capacity for colony formation in agarose show that LCL may acquire " malignant " characteristics in vitro. Changes of other phenotypic characteristics following karyotypic alterations have been reported previously. Glimelius et al. (1975) found an increased agglutinability with Concanavalin A. Nilsson et al. (1974) showed an altered amount of secreted and surface located B,-microglobulin and Nilsson and Ponten (1975) reported morphological alterations. The importance of using recently established LCL in comparative studies on any phenotypic characteristics is, ofcourse, underlined by the present and previous findings. The causative role of EBV in the heterophileantibody-positive infectious mononucleosis (IM) is firmly established (Henle et al., 1968; Niederman et al., 1968), However, the possible oncogenic properties of EBV are still obscure. Arguments for an oncogenic role are provided by sero-epidemiological studies (Henle et al., 1969, 1971) by the exclusive restriction of EBV-genomes to BL and nasopharyngeal carcinoma in all hybridization tests conducted so far and the concomitant presence of the EBNA antigen (zur Hausen et al., 1970; Nanoyama et al., 1973; Lindahl et al., 1974), by the capacity of EBV to " immortalize " B lymphocytes in vitro (for a review see Klein, 1975) and by the fatal lymphoproliferation caused by EBV upon injection into marmosets (Shope e l al., 1973; Miller, 1975; Werner et al., 1975; Falk et al., 1976) or owl monkeys (Epstein et al., 1973). The present study and earlier reports (Nilsson and Pontkn, 1975; Jarvis et al., 1974; Zech et al., 1976) favour the hypothesis of a progressive evolution of a BL cell clone where virus-induced transformation, a sequence of cytogenetic changes and a stepwise evolution of independence from host control mechanisms all interact in bringing about the final result. The genesis of BL would thus be similar t o what is probably occurring in most in vivo tumors-a progressive stepwise selection of the fittest single clone of tumor cells (Foulds, 1954; KIein and Klein, 1977). In the above reasoning it is assumed that only one and the same strain of EBV infects cells of both the LCL and BL types. So far no solid evidence for



the existence of various strains of EBV with the capacity to transform lymphocytes has been reported, but the possibility should be kept in mind that different substrains of the virus are causative in IM and BL. In the former disease infection in vivo would lead to diploid LCL precursor cells. In BL an interaction of vital information and genetic changes of the host cell genome and perhaps an impaired immune defense of the host are required for the development of a lymphoma.



This work was supported by the Swedish Cancer Society, the Stehlin Foundation for Cancer Research and Contract No. NO1 CB 33316 within the Virus Cancer Program of the National Cancer Institute. The skilful technical assistance of Mrs. Agneta Snellman, Mrs. Sun Ock Yim and Mr. Randall C. Shepard and the secretarial work of Mrs. Kerstin Lindberg is gratefully acknowledged.



Des lignies de cellules h6matopol6tlques humaines, qui o n t 6t6 class6es d’aprh I’6tude de param&res clonaux, morphologiques, chromosomlques e t fonctionnels en lignees lymphoblastoldes (LCL) d’origine probablement non n6oplasique e t en llgn6es lymphomateuses, my6lomateuses e t leuc6miques d’origine maligne avhree, ont et6 testees pour 6valuer leur potentiel t u m o r i g h e a p r h transplantation sous-cutanhe chez des souris nues” e t leur capacit6 de se d6velopper en milieu semi-solide in vitro. Les LCL r6cemment 6tablies ne se sont d6veloppies ni chez les souris ni dans I’agarose. Par contre, parml les LCL dans lesquelles des alt6rations chromosomlques secondaires Btaient apparues au cours d’une culture continue ayant dur6 plusieurs ann6es. quelques-unes Btaient t u m o r i g h e s ou pouvaient former des colonies dans I’agarose. La plupart des lign6es lymphomateuses ont form6 des colonies dans I’agarose e t des tumeurs chez les souris. L’une des deux lign6es de my6lome a produit une tumeur sous-cutanhe qui, cependant, pr6sentait une n6crose centrale. L’autre ne s’est pas d6velopp6e. Ces deux lignies ont form6 des colonies dans I’agarose. La llgn6e de leuc6mie my6loVde 6tait tumorighe, mais I’une des trois llgn6es de leuc6mie lymphocytaire test6es ne s’est pas d6velopp6e chez les souris. Touter les lign6es IeucQmiques o n t form6 des colonies dans I’agarose. Les conclusions tlr6es de cette 6tude sont les suivantes: 1) Des deux types de lign6es contenant le virus d’Epstein-Barr (LCL e t lign6es de lymphome de B u r k i t t (BL)), seules les lign6es B L forment des tumeurs lorsqu’on les inocule par voie sous-cutanbe h des souris nues” e t peuvent r e developper in vitro dans I’agarose. Ce n’est donc pas cause de l a transformation par I’EBV en elle-m6me que les lymphocytes deviennent necessairement t u m o r i g h e s chez les souris ‘‘ nues ”; Ils n’acqui8rent pas non plus l a capacite de former des colonies dans I’agarose. 2) Les modifications du caryotype e t de plusieurs caractbristiques ph6notyplques que I’on observe dans les LCL initialement diploldes pendant une culture prolong6e in vitro peuvent aller de pair avec I’acquisitlon de la capaciti de se d6velopper a p r h injection sour-cutanbe chez les souris “nues” e t dans I’agarose in vitro. 3) Les dlff6rences observ6es entre les lignees n6oplasiques, dont certaines r e d6veloppent chez les souris“ nues ” ou dans I’agarose e t d’autres pas, semblent indiquer qu’aucun de ces deux tests ne constitue un critare sGr pour determiner l a malignit6 des lignees de lymphome, de leuc6mie e t de my6lome humains. ‘@


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Tumorigenicity of human hematopoietic cell lines in athymic nude mice.

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