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.

338

NILSSON ET AL.

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.

TABLE I TUMORIGENICITY IN NUDE MICE A N D COLONY FORMATION IN AGAROSE BY HEMATOPOIETIC CELL LINES

Cell line

Type of line

a

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

U-704

LCL, new line

U-718

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

Jijoye

BL, old line

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

+ +

++ +

Reference

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)

Karyotypelreference

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 -

+

+-

++ + +

4-

+

+ +

339

TUMORIGENICITY OF LYMPHOID CELL LINES IN NUDE MICE TABLE I (Continued)

Colony Cell line

Type of line

a

gEtLe

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)

(1974)

Aneuploid/Zech et al. (1976)

+ +

Aneuploid/Ben-Bassat et al.

-

(1974) U-715 M

LL, old line

Nilsson and Sundstrom (1974)

u-937

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

zur Hausen (1972) Sundstrom and Nilsson

RPM18226

Myeloma, old line

Matsuoka et al. (1967)

DG-75

s

96

Ben-Bassat et al. (1977)

NT NT NT NT NT NT NT -

+

(1977)

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

(1976)

Aneuploid/Huang et al.

f6

(1969)

MOLT 4

Myeloma, old line ALL, old line

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

JM

ALL, old line

Schwenk and Schneider

U-266 B1

CCRF-CEM K 562

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.

+

MATERIAL AND METHODS

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.

TABLE 11

RESULTS

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.

RELATIONSHIP BETWEEN TYPE OF HEMATOPOIETIC LINE, TUMORIGENICITY, COLONY FORMATION IN AGAR A N D KARYOTYPE

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

f

~

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.

n

TUMORIGENICITY OF LYMPHOID CELL LINES IN NUDE MICE

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

341

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

342

NILSSON ET AL.

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.

TUMORIGCNICITC

ACKNOWLEDGEMENTS

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 LIGN~ES DE CELLULES HCMATOPOVGTIQUES C H E Z LES SOURIS ATHYMIQUES NUES”

HUMAINES

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. ‘@

REFERENCES

ADAMS,R. A., FOLEY, G. E., FARBER,S., FLOWERS,A., LAZARUS, H., and HELLERSTEIN, E. E., Serial transplantation of Burkitt’s tumor (EB3) cells in newborn Syrian hamsters and its facilitation to antilymphocyte serum. Cancer Res., 30, 338-345 (1970. ADAMS, R. A., HELLERSTEIN, E. E., POTHIER, L., FOLEY,G. E., LAZARUS, H., and STUART,A. B., Malignant potential of a cell line isolated from the peripheral blood in infectious mononucleosis. Cancer, 27, 651-657 (1971). ADAMS,R. A., POTHIER, L., HELLERSTEIN, E. E., and BOILEAU, G., Malignant immunoblastoma: immunoglobulin synthesis and the progression to leukemia in heterotransplanted acute lymphoblastic leukemia, chronic lymphatic leukemia, lymphoma, and infectious mononucleosis. Cancer, 31, 1397-1407 (1973). ARNSTEIN,P., TAYLOR, D. 0. N., NELSON-REES, W. A., E. H., Propagation of human HUEBNER, R. J., and LENNETTE, tumors in antithymocyte serum-treated mice. J. nat. Cancer Inst., 52, 71-84 (1974). B~CHET J., M., FIALKOW,P., NILSSON,K., and KLEIN,G., Immunoglobulin synthesis and glucose-6-phosphate dehydrogenase as cell markers in human lymphoblastoid cell lines. Exp. Cell Res., 89, 275-282 (1974). BELPOMME, D., MINOWADA, J., and MOORE,G. E.. Are some human lymphoblastoid cell lines established from leukemic

tissues actually derived from normal leukocytes ? Cancer, 30, 282-287 (1972). BEN-BASSAT, H., GOLDBLUM, N., MITRANI, S., GOLDBLUM, T., YOFFEY,J. M., COHEN,M. M., BENTWICH, Z., RAMOT,B., KLEIN. E.. and KLEIN. G.. Establishment in continuous culture of a new type of lymphocyte from a ‘‘ Burkitt-like ” malignant lymphoma (Line D.G.-75). I n / . J. Cancer, 19, 27-33 (1977). CHRISTOFINIS, G. J., Chromosome and transplantation results of a human leukocyte cell line derived from a healthy individual. Cancer, 24, 649-651 (1969). DEAL,D. R., GERBER, P., and CHISARI,F. V., Heterotransplantation of two human lymphoid cell lines transformed in vitro by Epstein-Barr virus. J. nut. Cancer Inst., 47, 771-780 (1971). EPSTEIN,M. A., Studies with Burkitt’s lymphoma. Wistar Inst. Symp. Monogr., 4, 69-82 (1965). EPSTEIN, M. A,, HUNT,R. D., and RABIN,H., Pilot experiments with EB virus in owl monkeys (Aotus hivirgatus). I. Reticuloproliferative disease in an inoculated animal. I n [ . J. Cancer, 12, 309-318 (1973). EPSTEIN,A. L., HERMAN,M. M., KIM, H., DORFMAN,R., and KAPLAN,H. S., Biology of the human malignant lymphomas. 111. Intracranial heterotransplantation in the nude, athymic mouse. Cancer, 37, 21 58-2176 (1976).

TUMORIGENICITY OF LYMPHOID CELL LINES IN NUDE MICE

EPSTEIN,A. L., and KAPLAN,H. S., Biology of the human malignant lymphomas. I. Establishment in continuous cell culture and heterotransplantation of diffuse histiocytic lymphomas. Cancer, 34, 1851-1872 (1974). FAGRAEUS, A., NILSSON,K., LIDMAN, K., and NORBERG, R., Reactivity of smooth-muscle antibodies, surface ultrastructure, and mobility in cells of human hematopoietic cell lines. J. nat. Cancer Inst., 55, 783-789 (1975). FALK,L., DEINHARDT, F., WOLFE,L., JOHNSON, D., HILGERS, J., and D E - T H G., ~ , Epstein-Barr virus: experimental infection of callithrix jacchus marmosets. Int. J. Cancer, 17, 785-788 (1976).

FIALKOW, P. J., KLEIN,G., GARTLER, S. M., and CLIFFORD, P., Clonal origin for individual Burkitt tumours. Lancet, 1, 384-386 (1970).

FOGH,J., and FOGH,H., A method for direct demonstration of pleuropneumonia-like organisms in cultured cells as judged by orcein staining. Proc. SOC. exp. Biol. ( N . Y . ) , 117, 899-901 (1964).

FOLEY,G. E., LAZARUS,H., FARBER,S., UZMAN,B. G., BOONE,B. A., and MCCARTHY,R. E., Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer, 18, 522-529 (1965). FOULDS,L., The experimental study of tumor progression: a review. Cancer Res., 14, 327-339 (1954). FREEDMAN, V. H., and SHIN, S., Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell, 3, 355-356 (1974). GIOVANELLA, B. C., and STEHLIN, J. S., Heterotransplantation of human malignant tumors in “ nude ” thymusless mice. I. Breeding and maintenance of nude mice. J. nat. Cancer Inst., 51, 615-619 (1973).

343

and Burkitt lymphoma. J. nat. Cancer Inst., 43, 1129-1146 (1969).

HUBER, Ch., SUNDSTROM, C. C., NILSSON,K., and WIGZELL, H., Surface receptors o n human haematopoietic cell lines. Clin. exp. Immunol., 25, 367-378 (1976).

IMAMURA, T., HUANG, C. C., MINOWADA, J., and MOORE, G. E Heterologous transplantation of human hematopoietic cell lines. Cancer, 25, 1320-1331, (1970). IMAMURA, T., and MOORE,G. W., Ability of human hematopoietic cell lines to form colonies in soft agar. Proc. SOC. exp. Biol. ( N . Y . ) , 128, 1179-1183 (1968). JARVIS, J. E., BALL,G., RICKINSON, A. B., and EPSTEIN, M. A., Cytogenetic studies on human lymphoblastoid cell lines from Burkitt’s lymphomas and other sources. In/. J. Cancer, 14, 716-721 (1974).

KLEIN,G., The Epstein-Barr virus and neoplasia. New Eng. J. Med., 293, 1353-1357 (1975). KLEIN,G., DOMBOS,L., and GOTHOSKAR, B., Sensitivity of Epstein-Barr virus (EBV) producer and non-producer human lymphoblastoid cell lines t o superinfection with EB-virus. In/. J. Cancer, 10, 44-57 (1972). KLEIN,G., GIOVANELLA, B., WESTMAN, A., STEHLIN,J., and MUMFORD,D., An EBV-genome-negative cell line established from an American Burkitt lymphoma: receptor characteristics, EBV infectability and permanent conversion into EBV-positive sublines by in vitro infection. Intervirology, 5, 319-334 (1975).

KLEIN,G., and KLEIN,E., Rejectability of virus induced tumors and non-rejectability of spontaneous tumors. A lesson in contrasts. Transplant. Proc., in press. KLEIN,G., LINDAHL, T., JONDAL,M., LEIBOLD, W., NILSSON, K., and SUNDSTROM, C., Continuous lymphoid cell lines with B-cell characteristics that lack the Epstein-Barr virus genome, derived from three human lymphomas. Proc. Nut.

GIOVANELLA, B. C., and STEHLIN, J. S., Assessment of th: malignant potential of cultured cells by injection in “ nude mice. In: Rygaard J. (ed.), Proc. 1st I n / . Workshop on nude Acad. Sci. (Wash.), 71, 3283-3286, (1974). mice, Aarhus 1973, pp. 279-284, Fischer, Stuttgart (1974). KLEIN,G., ZEUTHEN, J., TERASAKI, P., BILLING, R., HONIG,R., JONDAL,M., WESTMAN, A,, and CLEMENTS, GIOVANELLA, B. C., STEHLIN,J. S., and WILLIAMS, L. J., G., Inducibility of the Epstein-Barr virus (EBV) cycle and surface marker Heterotransplantation of human malignant tumors in properties of EBV-negative lymphoma lines and their in vitro ‘‘ nude ” thymusless mice. 11. Malignant tumors induced by EBV-converted sublines. In press. injection of cell cultures derived from human solid tumors. J. frat. Cancer Inst., 52, 921-930 (1974). LEVIN,A. G., FRIBERG JR., S., and KLEIN,E., XenotransGIOVANELLA, B. C., YIM,S. O., STEHLIN, J. S., and WILLIAMS, plantation of a Burkitt lymphoma culture line with surface immunoglobulin specificity. Nature (Lond.), 222, 997-998 L. J., Development of invasive tumors in the ‘‘ nude ” mouse (1 969). after injection of cultured human melanoma cells. J. nat. Cancer Inst., 48, 1531-1533 (1972). LINDAHL, T., KLEIN,G., REEDMAN, B. M., JOHANSSON, B., and SINGH,S., Relationship between Epstein-Barr virus (EBV) GLIMELIUS, B., NILSSON, K., and PONTBN,J., Lectin agglutinD N A and the EBV-determined nuclear antigen (EBNA) ability of non-neoplastic and neoplastic human lymphoid in Burkitt lymphoma biopsies and other lymphoproliferative cells in vitro. Int. J . Cancer, 15, 888-896 (1975). Int. J. Cancer, 13, 764-772 (1974). malignancies. GRAVELL, M., LEVINE, P. H., MCINTYRE, R. F., LAND,V. J., LOZZIO,C. B., and LOZZIO,B. B., Human chronic myeloand PAGANO,J. S., Epstein-Barr virus in an American patient with Burkitt’s lymphoma: detection of viral genome geneous leukemia cell line with positive Philadelphia chromoin tumor tissue and establishment of tumor-derived cell some. Blood, 45, 321-334 (1975). line (NAB). J. nat. Cancer Inst., 56, 701-704 (1976). MACPHERSON, I., and MONTAGNIER, L., Agar suspension HAYFLICK, L., and CHANOCK, R. M., Mycoplasma species culture for the selective assay of cells transformed by polyoma in man, B a r / . Rev., 29, 185-221 (1965). virus. Virology, 23, 291-294 (1964). HENLE,G., HENLE.W., CLIFFORD, P., DIEHL,V., KAFUKO, MANOLOV,G., and MANOLOVA, Y., Marker band in one G. W., KIRYA,B. G., KLEIN,G., MORROW,R. H., MUNUBE, chromosome 14 from Burkitt lymphomas. Nature (Lond.), P. M., and ZIEGLER, J. L., Anti237, 33-34 (1972). G. M. R., PIKE,P., TUKEI, bodies t o Epstein-Barr virus in Burkitt’s lymphoma and MATSUOKA, Y., MOORE,G. E., YAGI,Y., and PRESSMAN, D., control groups. J. nut. Cancer Inst., 43, 1147-1157 (1969). Production of free light chains of immunoglobulin by a hematopoietic cell line derived from a patient with multiple HENLE,G., HENLE,W., and DIEHL,V., Relation of Burkitt’s myeloma. Proc. Soc. exp. Biol. ( N . Y . ) , 125, 1246-1250 tumor-associated herpes type virus to infectious mononu(1967). cleosis. Proc. nut. Acad. Sci. (Wash.), 59, 94-101 (1968). HENLE, G., HENLE,W., KLEIN,G., GUNVEN, P., CLIFFORD, P., MILLER,G., Th e oncogenicity of Epstein-Barr virus. J. MORROW,R. H., and ZIEGLER,J., Antibodies to early infect. Dis., 130, 187-205 (1974). Epstein-Barr virus induced antigens in Burkitt’s lymphoma. MINOWADA, J., NONOYAMA, M., MOORE,G. E., RAUCH,A. M. J. nat. Cancer Inst., 46, 861-871 (1971). and PAGANO,J. S., The presence of the Epstein-Barr viral HUANG,C. C., Hou, Y., WOODS,L. K., MOORE,G. E., and genome in human lymphoblastoid B-cell lines and its abMINOWADA, J., Cytogenetic study of human lymphoid T-cell sence in a myeloma cell line. Cancer Res., 34, 1898-1903 lines derived from lymphocytic leukemia. J . nut. Cancer (1974). Inst., 53, 655-660 (1974). MINOWADA, J., OHNUMA, T., and MOORE,G. E., RosetteHUANG,C. C., IMAMURA, T., and MOORE.G . E., Chromoforming human lymphoid cell lines. I. Establishment and somes and cloning efficiencies of hematopoietic cell lines evidence for origin of thymus-derived lymphocytes. J. nut. Cancer Inst., 49, 891-895 (1972). derived from patients with leukemia, melanoma, myeloma

344

NILSSON ET AL.

REEDMAN, B. M., and KLEIN,G., Cellular localization of an Epstein-Barr virus (EBV) associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. I n t . J. Cancer, 11, 499-520 (1973). RYGAARD, J., Immunobiology of the mouse mutant “ nude ”. Acta Path. Microbiol. Scand., 77, 761 -762 (1969). RYGAARD, J., and POVLSEN, C. D., Heterotransplantation of a human malignant tumor to “ nude ” mice. Acta Path. Microbial. Scand., 77, 758-760 (1969). SCHWFNK, H.-U., and SCHNEIDER, U., Cell cycle dependency o f a T-cell marker on lymphoblasts. Blur, 31, 299-306 (1975). SHOPE, T., DECHAIRO, D., and MILLER,G., Malignant lymphoma in cotton-top marmosets after inoculation with Epstein-Barr virus. Proc. nut. Acad. Sci. ( W a s h . ) , 70. 2487-2491 ( I 973). SOUTHAM, C. M., BURCHENAL, J. H., CLARKSON, B.,TANzI,A., MACKEY,R., and MCCOMB,V., Heterotransplantation of human cell lines from Burkitt’s tumors and acute leukemia into newborn rats. Cancer, 23, 281-299 (1969~). SOU,rHAM, c. M., BURCHENAL,J. H., CLARKSON, B., TANZI, A, MACKEY, R., and MCCOMB,V., Heterotransplantability of human cell lines derived from leukemia and lymphomas into immunologically tolerant rats. Cancer, 24, 21 1-222 (19696). STILES,C. D., DESMOND, W., CHUMAN, L. M., SATO,G., NILSSON, K., BENNICH, H., JOHANSSON, S. G. O., and PONTEN, and SAIER,Jn. M. H., Growth regulation of heterologous tissue culture cells in the congenitally athymicnude mouse. I I . J., Established immunoglobulin producing myeloma (IgE) Relationship of growth behavior in vitro to tuniorigenicity. and lymphoblastoid (IgG) cell lines from an IgE myeloma Cancer Res., 36, 1353-1356 (1976). patient. Clin. exp. Immiinol., 7, 477-489 (1970). SUNDSTROM, C., and NILSSON, K., Establishment and characNILSSON,K., EVRIN,P.-E., and WELSH,K . I., Production terization of a human histiocytic lymphoma cell line (U-937). of pz-microglobulin by normal and malignant human cell In!. J. Cancer, 17, 565-577 (1976). lines and peripheral lymphocytes. Transplant. Rev., 21, 53-84 (1974). TOSHIMA, S., TAKAGI, N., MINOWADA, J., MOORE,G. E., and SANDBERG, A. A., Electron microscopic and cytogenetic NILSSON,K., and P O N T ~ N J.,, Classification and biological studies of cells derived from Burkitt’s lymphoma. Cancer nature of established human hematopoietic cell lines. Int. J . Res., 27, 753-771 (1967). Cancer, 15, 321-341 (1975). WERNER, J., WOLF, H., APODACA, J., and zur HAUSEN, H., NILSSON, K., and SUNDSTROM, C.. Establishment and characLymphoproliferative disease in a cotton-top marmoset after teristics of two unique cell lines from patients with lymphoinoculation with infectious mononucleosis-derived Epsteinsarcoma. I n t . J . Cancer, 13, 808-823 (1974). Barr virus. Int. J. Cancer, 15, 1000-1008 (1975). PANTELOURIS, E. M., Absence of thymus in a mouse mutant. YATA,J., and KLEIN,G., Some factors affecting membrane Nature (Lond.), 217, 370-371 (1968). immunofluorescence reactivity of Burkitt lymphoma tissue culture lines. l n t . J . Cancer, 4, 767-775 (1969). P O N T ~ NJ., , Spontaneous lymphoblastoid transformation of long-term cell cultures from human malignant lymphoma. ZECH, L., HAGLUND,U., NILSSON,K.. and KLEIN,G., Int. J . Cancer, 2, 31 1-325 (1967). Characteristic chromosal abnormalities in biopsies and lymphoid-cell lines -from patients with Burkitt and nonP O N T ~ NJ.,, and MACINTYRE, E. H., Long term culture of normal and neoplastic human glia. Acta Path. Microbiol. Burkitt lymphomas. Int. J. Cancer, 17. 47-56 (1976). Stand., 74, 465-486 (1968). Z U R HAUSEN, H., DIEHL,W., WOLF,H., SCHULTE-HOLTHAUSEN, H., and SCHNEIDER, U . , Occurrence of Epstein-Barr POVLSEN, C. 0..and RYGAARD, J., Heterotransplantation virus genomes in human lymphoblastoid cell lines. Nature of human adenocarcinomas of the colon and rectum to the New Eiol., 237, 189-190 (1972). mouse mutant nude. A study of nine consecutive transplantations. Acla Path. Microbial. Scand., Sect A , , 79, 159-169 Z U R HAUSEN,H., SCHULTE-HOLTHAUSEN, H . , KLEIN,G., (1971). HENLE,W., HENLE,G., CLIFFORD, P., and SANTFSSON, L., EBV D N A in biopsies of Burkitt tumours and anaplastic PuLvEnTAFT, R. J. V., A study of malignant tumours in carcinomas of the nasopharynx. N a t i v e (Land.), 228, Nigeria by short term tissue culture. J. d i n . Path., 18, 261-273 1056-1058 (1970). (1965).

MOORE, G. E., and GERNER,R. E., Cancer immunityhypothesis and clinical trial of lymphocytotherapy for malignant diseases. Ann. Surg., 172, 733-739 (1970). NADKARNI, J. S., NADKARNI, J. J., CLIFFORD, P., MANOLOV, S., FENYO,E. M., and KLEIN,E., Characteristics of new cell lines derived from Burkitt lymphomas. Cancer, 23, 64-79 (1969). NANOYAMA, M., HUANG,C. H., PAGANO,J. S., KLEIN,G., and SINGH,S., DNA of Epstein-Barr virus detected in tissue. Proc. nat. Acad. Sci. ( W a s h . ) , 70, 3265-3268 (1973). NANOYAMA, M., and PAGANO,J. S., Detection of EpsteinBarr viral genome in non-productive cells. Nature New Biol., 233, 103-106 (1971). NIEDERMAN, J. C., MCCOLLUM,R. V., HENLE,G., and HENLE, W., Infectious mononucleosis: clinical manifestations in relation to EB virus antibodies. J . Amer. med. Ass., 205, 205-209 ( I 968). NILSSON,K., High-frequency establishment of human immunoglobulin producing lymphoblastoid lines from normal and malignant lymphoid tissue and peripheral blood. Int. J. Cancer, 8,432-442 (19710). NILSSON, K., Characteristics of established human myeloma and lymphoblastoid cell lines in long term culture. Int. J . Cancer, 7, 380-396 (1971b).