Znt. J. Cancer: 16, 74-82 (1975)
DISTINCTIVE BANDED MARKER CHROMOSOMES OF HUMAN TUMOR CELL LINES
Walter A. NELSON-REES, Robert R. FLANDERMEYER and Paula K. HAWTHORNE Cell Culture Laboratory, University of California, School of Public Health, Naval Biomedical Research Laboratory, Oakland, California, 9462S, USA
Nine human tumor cell lines (five breast carcinomas and four sarcomas) have been studied and each revealed groups of distinctive banded marker chromosomes which can serve to identify them and aid in monitoring cell line specificity. This was possible neither by conventional karyology in terms of numbers and morphology of chromosomes nor by glucose-6-phosphate-dehydrogenasemobility which was type B for all cultures. The SigniJcance of the clonal nature of the cell lines is discussed.
We recently detected HeLa-cell contamination of human cell cultures being utilized in several laboratories in this country and abroad (NelsonRees et al., 1974a, b). Our results on two cell lines, MA160 and H. Ep. -2, were confirmed by similar methods (Zalta et al., 1974; Lin and Goldstein, 1974). Our conclusions were based primarily on combined results obtained by two different methods with which previous workers had revealed HeLa cell-like characteristics, namely type A (fast) isoenzyme mobility pattern for glucose-6-phosphate dehydrogenase, G6PD (Gartler, 1966) and multiple banded marker chromosomes (Miller et al., 1971). The frequency in the general population of individuals with G6PD type A is relatively low (see Kirkman, 1971, for review) and one expects that only a fraction thereof develop neoplasms. Biopsies of only a few tumors are placed in culture and not all cultures grow up. It follows that the majority of cell lines in existence are of the type B (slow) mobility G6PD. Of these, many have achieved remarkable growth potential and, like HeLa, are widely used in many laboratories. Potential for contamination among such human, non-HeLa cultures therefore also exists, but we believe could be readily detected. We Received: March 10, 1975.
74
have selected a number of tumor cell lines exhibiting G6PD type B and have studied their chromosomes after Q and G banding. Unique groups of abnormal chromosomes (" markers ") were found in each cell line which can serve to distinguish them from each other and aid in monitoring their specificity. MATERIAL A N D METHODS
Cell lines Table I lists for each cell line studied the type of tumor of origin, the designation of the line derived from it, our immediate source and a published reference for each line. Cell culture method and chromosome analysis
All cells were grown as previously described (Nelson-Rees et a/., 19746). Methods for chromosome analysis were detailed previously (Nelson-Rees et a[., 1974a, b). Isoenzyme analysis The isoenzyme mobility pattern for G6PD was determined by Dr. W. D. Peterson, Jr., following his methods (Peterson et al., 1968).
4
VI
46-59
0
See addendum.
6
As human tumor cell culture re-isolated from cat (Nelson-Rees et al., 1972).
As recloned clone.
(Rasheed et al., 1974)
Fibrosarcoma HTlO80
S . Rasheed
0
0
late passage
* As clonal cultures.
a
11-27
?
9-1 7
62
33
3-26 '
18-39
124-128
Y
0
8-14
Y
6-10
18-31
P
0
24-42
Y
0"
T. O'Connor and G. Shibley (Chan et a / . , 1974)
R. McAllister (McAllister el a/., 1972)
R. McAllister (McAllister et al., 1971) J. Rhirn (Rhim et al., 1975)
E. Lasfargues (Lasfargues and Ozello, 1958) E. Lasfargues (Reed and Gey, 1962) M. Brennan (Soule et al., 1974) C. McGrath (McGrath ef al., 1974a) R. Cailleau (Young et al., 1974) G. Lowell (Fogh and Trernpe, 1975)
early passage
Liposarcoma SA, (strain B)
Rhabdomyosarcoma RDll4
R970 (derivative of Te85 subline F)
Osteosarcoma Te85 subline F
SKBR-3
MCF-7 (derivative of 734B) MDA-MBl57
734B
ALAB 496
Mammary carcinoma BT-20
TABLE I
44-48
59-65
51-67
43-52
51-53
50-59
46
64
66
46, 48
51
54
83
62, 64
51-68 75-102
87
74
54
49
79-89
68-76
53-57
43-53
TUMOR CELL LINES WITH G6PD TYPE B A N D DISTINCT MARKER CHROMOSOME PATTERN
+
+ translocated + translocated
-
-
-
-
-
-
-
-
-
v)
5
c
r
Gr
B
2
9
q
E
2
=!
0
3
U
t;
NELSON-REES ET AL.
RESULTS
Conventional staining methods demonstrated previously that morphologically aberrant chromosomes existed in these cell lines, but banding delineated and specified with greater precision each marker chromosome. Thus, for example, RDI 14 (Nelson-Rees et a/., 1972) had earlier revealed morphologically aberrant chromosomes including a ring, and HT1080, pseudodiploidy with aberrant chromosomes (Rasheed et a/.. I974), but banding demonstrated the specific origin of the “ marker ” chromosomes through rearrangement of normal chromosome banding patterns (Fig. I ) . For the present study, we first established the range and the modal number ofchromosomes in at least 20 metaphases of each cell culture (Table I). These figures alone could not serve to distinguish clearly between the different cell lines.
Q banding in at least 20 metaphases per culture revealed, as expected, absence of a Y chromosome in female cells and its presence in male-derived cells (Table 1). As previously noted (Nelson-Rees rt a/., 1974a), SA4 (strain B) cells exhibited an autosome-Y translocation. This chromosome is now designated as “ marker ” number 4 (M4) of this cell line (Fig. I). We studied at least 15 metaphases and constructed at least five karyotypes per cell culture following trypsin-Giemsa banding. All cell lines revealed groups of marker chromosomes as demonstrated by rearrangement of normal, human banding configurations. We concentrated o n all markers, up to a maximum of five, which were seen in all metaphases of individual cell lines at different passage levels (Fig. I ) . The five breast carcinoma cell lines exhibited the longest marker chromosomes observed. Although they were each clearly derived from different chromosomes (Table 11) the production of these long chromosomes most often involved most or all of the long arm of either chromosomes No. I , 2, or 3, the longest chromosomes in the normal karyotype. In two instances, cultures of the same line, but in use in different laboratories and passage levels, were studied and found to have identical markers. First, a culture of 734B was received at passage 3, grown and studied in our laboratory in 1972. The present marker chromosomes were from that culture.
76
Cultures of these cells were the subject of the work of Soule et a / . (1974). From the latter was derived a strain designated MCF-7 which is currently the subject of extensive research in viral oncology (McGrath c’f a/., 1974~7,b) l . MCF-7 was studied at passage 124 and banded in our laboratory in October, 1974. Both 734B and MCF-7 have a complex of identical marker chromosomes. Second, MDA-MB-157 cells at passage 6 were received and studied in our laboratory in May, 1973, prior to being described in the original publication (Young C? a/., 1974). Several markers studied in our culture are obviously identical to those appearing, but not described, in passage 3 cells i n that publication (6t.g. our No. I marker is indicated in the publication by an arrow). Among the sarcoma-derived cells another dual example is described. The osteogenic sarcomaderived Te85 cell line (McAllister et a/., 1971) was initiated and sublines established in 1969. The chromosomes of subline F shown are from passage 12. A derivative of this subline was recent I y u t i I ized in t ran sfornia t ion experiments with the Kirsten strain of murine sarcoma virus (Rhim et a/., 1975). The cells of the latter culture, designated R970, I8 passages after transformation, have markers identical to those of Te85 subline F. The same markers were observed (data not shown) in R970 cells when recovered following inoculation into *‘ nude ” mice where they induced tumors (Rhim et a/., MS submitted). SA4 (strain B) has been studied over many passages (Chan rt a/., 1974; Shibley et al., 1974; Bandyopadhyay et a/., 1974). In addition to the Y-autosome translocation mentioned above, we report a complex of marker chromosomes seen first at “ early ” and then at “ late ” passages following several clonings of the culture. However, one large submetacentric marker (M5) possibly not present in dl uncloned cells, was excluded from the culture in the course of cloning, but a complex of markers, including the Y-autosome translocation, persists. Cells of HT1080 received in 1972 at passage 8 and initially described by us as being pseudo-
* Drs. C. McGrath and M. Rich announced at the Xlth International Cancer Congress at Florence Congress that thecell line would be designated MCF-7 and the virus isolated from i t . 734.
FIGURE 1 Marker chromosomes observed in all cells of each cell line or its derivative. The proposed origins of banding patterns through rearrangements of normal human chromosomes are described in Table 11. Note: Cells of line SA4 (strain B) do not have a normal Y chromosome. Therefore, marker, number 4 of -‘early ” and ‘‘ late ” passages is described with the aid of a Y chromosome taken from a normal Y karyotype.
00
4
late *'
'
No. 1 1 with addition on q of portion of No. 3 q.
No. 5 with addition on p
of portion of No. 17 q.
7
Same
Same
No. 1 deletion of portion of p near centromere.
Same No. 9 with addition on q of portion of No. 8 q.
No. 16 with addition on p No. 17 with addition on p Small ring (on description). of portion of No. 14 q. No. 3 with addition on No. 21 with addition on p Y with addition on q of p of portions of No. 9 q portion of No. 15 q. of portion of No. 1 1 q. and No. 13 q. No. 9 with addition Same Same on q of portion of No. 13 q. of portion of No. 10 q.
No. 9 with deletion of p and addition on q of portion of No. 8 q,
No. 19 with addition on q of portion of No. 1Oq. No. 21 with addition on p of portion of No. 8 p.
No. 19 with addition on q of portion of No. 6 p.
No. 2 with addition on q of portion of No. 17 q (or X P).
M5
long arm of normal chromosome (Chicago Conference, 1966). Resembles HeLa No. 4 marker (see Nelson-Rees el a/.,19740) difference being that HeLa No. 4 has portions of weakly staining No. 22 and heavy bands of NO. I2 q
**
Same No. 5 with addition on q of portion No. 3 p. lsochromosome of No. I O q .
p = short arm; q
HT1080
*.
SAI *' early
Te8 5 (subline F) R970 RDI 14
of portions of No. 8 q and 2 q.
Same
No. 13 with addition on p No. 5 with addition on q of portion of No. 1 q. of portion of No. I q.
Same
No. 12 with addition on p No. 8 with addition on q of portions of No. 18 q and No. 2 q. No. 1 I with addition on q No. 7 with addition on q of portion of No. 5 q. of portion of No. 7 q.
Isochromosome of No. 2 q.
No. I 1 with deletion of portion of p.
of portion of No. 2 p.
No. 8 with addition on q
M4
No. 20 with addition on q No. 7 with addition on p of portion of No. 4 q. of portion of No. 10 q.
No. 14 with addition on p of portions of No. 18 q and No. 2 q. No. 12 with addition on q of portion of No. 12 q.
M3
SKBR-3
No. 3 p and centrornere with portion of No. 6 q.
No. 20 with addition of portions of No. 4 q and No. 12q. Same
No. 9 with addition on p of No. 4 q and most of No. 15 q.
No. 1 with addition on p of portion of X p.
M2
No. 2 with addition on q of portions of 5 q and 21 q.
Most of No. 1 1 with portions of No. 12 q and No. 5 q. No. 1 with addition on p of portion of No. 15 q (or X P). No. 20 with addition of portions of No. I 5 q and No. 1 q. Same
MI
MDA-MBI57
MCF-7
734B
ALAB
BT-20
Designation
DESCRIPTION OF POSSIBLE ORIGIN OF MARKER CHROMOSOMES IN HUMAN TUMOR CELL LINES
TABLE 11
DISTINCTIVE CHROMOSOMES OF TUMOR CELL LINES
diploid (Rasheed et al., 1974) were subsequently banded by Dr. W. F. Benedict at low and higher passage levels. Both passage levels carry markers presented here. DISCUSSION
Gartler’s hypothesis (Gartler, 1966) that many long-term human cell lines were, in fact, HeLa cells because, like HeLa, they exhibited the relatively rare type A mobility for G6PD, raised the question of whether prolonged cultivation of any human cell might not ultimately produce type A mobility (“convergence ”1. Some workers (Peterson et al., 1971) subsequently showed, however, that this was not necessarily the case. At least one cell line, Det 562, retained type B (slow) mobility throughout all passages in cultivation, ranging from 8 to 91. Our recent publications on cellular contamination (Nelson-Rees et al., 1974a, b) have again raised a similar question, namely whether all cell lines in cultivation for longer periods of time would not all acquire the same chromosome banding patterns as those demonstrated for HeLa and related cultures. We believe that the present results indicate that many cell lines, some now in long-term culture, retain their G6PD mobility and are unique entities, marked by peculiar and often complex chromosome banding patterns unrelated to those characteristic for HeLa cells. Undoubtedly, chance rearrangement alone could bestow upon any cell a “ marker ” chromosome similar or identical to one in another cell line, but the chances are minimal that entire complexes of multiple chromosome rearrangements as well as immunological characteristics would ultimately “ converge ”. Earlier data, now viewed in retrospect, conform to this concept. The first published description of a long-term cultivated human cell line with a unique banded chromosome marker and not considered to be of HeLa origin was, we believe, that by Miller et al. (1971) of WI-L2. This was a presumptive diploid lymphoblastoid line Dr. Benedict originally karyotyped the HT1080 cells by banding techniques and kindly informed us of his results (1973). We subsequently studied earlier passage material (see addendum in Rasheed el a/.,1974).
which was found to be pseudodiploid and carrying a Y-chromosome. Although we have not done trypsin-Giemsa banding on cells with that designation, a clonal derivative thereof also obtained from Dr. R. A. Lerner was studied by us several years later (Arnstein ei al., 1974) and found to show type B mobility for G6PD, also a Y-chromosome and still a modal number of 46 chromosomes. Similarly, Sykes’ ME180 cells of human cervical carcinoma (Sykes et al., 1970) as depicted, but not described, by Bordelon and Stubblefield (1974) showed no HeLa-like markers; our results (Arnstein et al., 1974) on cells with that designation received directly from Dr. J. A. Sykes indicated G6DP type B and a similar range in chromosome numbers. Elliot et al. (1974) described some G and C banded rearrangements in a line originating from a transitional cell carcinoma of the urinary tract. While the type of G6PD mobility is not indicated and the particular rearrangements are not defined, markers presented are, nevertheless, unique and may continue to serve to identify this cell line and its derivatives. One human report also utilizes banding patterns of marker chromosomes to distinguish a “ cell line ” from HeLa cefIs which it has been suspected of being for many years. H.Ep.-2 cell cultures from different sources have been indicted as HeLa cell contaminants (Gartler, 1966; Lin and Goldstein, 1974; Nelson-Rees et al., 19746). The work of Sinha and Pathak (1973) raises some doubt because it is not absolutely certain whether or not their results can be interpreted as describing two distinct strains of HeLa o r H.Ep-2 and HeLa as separate entities. It is possible that the small number of cells (4.5%) in the H.Ep.-2 culture which bore a Y chromosome were truly H.Ep.-2, but alternatively they may have been non-H.Ep.-2, non-HeLa contaminants. Of these, some might have also lost their Y. Nevertheless, those cells studied which lacked the Y “ h a d many features in common with the HeLa cells, including the st marker chromosomes ”. It would appear that additional immunological and enzyme data could serve to distinguish further between these sub-populations of the purported H.Ep.-2 cell culture and the HeLa culture, in order to distinguish between ‘‘ similarities ” and dissimilarities ” which in turn could either conform to or disagree with ‘‘ convergence. ” ‘I
79
NELSON-REES ET AL.
That contamination could be mistaken for ” or even for very convincing similarity between isolates of two separate tumors from different individuals was recently suggested by the use of banding techniques. We studied (Nelson-Rees, unpublished observations, 1975) two cell lines recently derived from adenocarcinomas of human colon and rectum (Tompkins et al., 19741, and sent to us by the originator. Both cell lines were G6PD type B and their cells carried a Y chromosome and showed aneuploidy involving the same chromosomes, as originally proposed by conventional staining. Neither, however, exhibited banded marker chromosomes (indicating re-arrangements) which could serve to distinguish these lines from each other. Pending results of further tests to define their identity, we presume as the originator now does (Tompkins, personal communication, 1975) that the two cell lines are one and the same. Clonal origin has been described for some, but not all, human tumors (see Kirkman, 1971 for review and Fialkow et al., 1972). It is tempting to ascribe multiple chromosome marker patterns specific for each cell line to unique events leading to formation of the individual tumors from which the cell lines were derived. Although our results conform to this concept, the distinct marker patterns may alternatively reflect “ stemcell line” (Levan, 1973) evolution and strict selection known to occur early in both tumorderived cultures and among long-term cultivated normal cells. Nevertheless, we have no direct chromosome data from the tumor specimens from which these lines were derived.
’‘ convergence
Like clones of HeLa cells or strains thereof maintained in separate laboratories, the cells studied here and other newer lines will acquire new markers through further rearrangements, or lose an occasional marker, naturally or due to cloning. However consistent monitoring and use of these markers as indicators will, we believe, permit identification of the cell lines and avoid cell contamination by newly established cell lines. AI>DENIlUM
After submitting this manuscript, we studied one additional culture from each of two lines at different passage levels and from other laboratories than those previously referred to in the body of the manuscript. BT-20 cells received from Dr. C. D. Aldrich at passage 274 had G6DP type B and clearly revealed marker chromosomes described here. SKBR-3 cells obtained from Dr. J. Fogh at passage 24 were also of type B G6PD mobility and at once revealed markers defined here. These results further confirm the value of banded marker chromosomes as persistent features of long-term cultivated cells. ACKNOWLEDGEMENT
We thank Dr. W. D. Peterson, Jr. for the isoenzyme mobility tests and Mr. I . P. Madin for assistance in karyology. This work was supported by Contract E 73-2001-NOl-CP-3-3237 in the Virus Cancer Program, National Cancer Institute, National Institutes of Health.
CHROMOSOMES MARQUEURS DlSTlNCTlFS DANS LES LIGNEES DE CELLULES TUMORALES HUMAINES Neuf ligiiL;rs de cellules tunlorales humaities ( 5 cancers du seiri ef 4 sarcotnes) otit Lte c5trrdiees; chacune contenait des groupes de chrotiiosonies marqueurs distinctifs que l’on a rep6rc;s en utilisant la technique des barides et qui peuvent servir a identifier les lignles cellulaires et aider contrder leur spkcificiti, ce qiri n’est pas possible par la caryologie classique, c’est-a-dire d’aprks le tiombre et la niorphologie rles chromosomes, ni d’aprks la tnobiliti de la glucose-6-phosphate-d~shydrogina.se, yiri etait de type B pour toutes les euftures. Les auteurs atialysent I’itnportunce cle la nature clorialc cles lignivs cellirlaires.
80
DISTINCTIVE CHROMOSOMES OF TUMOR CELL LINES
REFERENCES
ARNSTEIN, P., TAYLOR, D. 0.N.,NELSON-REES, W. A., MCALLISTER, R. M., NICOLSON, M., GARDNER, M. B., E. H. Propagation HUEBNER, R. J., and LENNETTE, RONGEY,R. W., RASHEED, S., SARMA,P. S., of human tumors in antithymocyte serum-treated HUEBNER, R. J., HATANAKA, M., OROSZLAN, S., mice. J. nut. Cancer Inst., 52, 71-84 (1974). GILDEN,R. V., KABIGTING, A., and VERNON, L., C-type virus released from cultured human rhabBANDYOPADHYAY, A. K., SHIBLEY,G. P., and domyosarcoma cells. Nature New Biology, 235, O'CONNOR,T. E., Characterization of an RNA3-6 (1972). dependent DNA-polymerase (RDDP-like) enzyme from an established human liposarcoma. Proc. MCGRATH,C. M., GRANT,P. M., SOULE,H. D., XIth International Cancer Congress, Florence, GLANCY, T., and RICH, M. A., Replication of Italy, October 20-26, 1974, oncorna-virus-like particle in human breast carcinoma cell line, MCF-7. Nature (Lond.), 252, BORDELON, M. R., and STUBBLEFIELD, E. Human 247-250 (1974a). tumours in mice confirmed by chromosomal analysis. Nature (Lond.) 252, 324-326 (1974). MCGRATH,C., GRANT,P., SOULE,H. D., and RICH,M. A., Characterization of an oncornavirusCHAN, E. W., SCHIOP-STANSLEY, P. E., and like particle replicating in a human breast. Proc. O'CONNOR,T. E., Isolation and characterization XIth International Cancer Congress, Florence, of an oncornavirus from a cell culture established Italy, October 20-26 (19746). from a human liposarcoma. Proc. Xlth International Cancer Congress, Florence, Italy, October MILLER, 0. J., MILLER,D. A., ALLDERDICE, P. W., 20-26, 1974. DEV, V. G., and GREWAL,M. S., Quinacrine fluorescent karyotypes of human diploid and CHICAGOCONFERENCE (I 966), Standardization in heteroploid lines. Cytogenetics, 10, 338-346 (1971). human cytogenetics. Birth defects. Original Article NELSON-REES, W. A., FLANDERMEYER, R. R., and Series, Vol. 1, p. 2, The National Foundation, New York (1966). HAWTHORNE, P. K., Banded marker chromosomes as indicators of intraspecies cellular contamination. ELLIOT, A. Y., CLEVELAND, P., CERVENKA, J., Science, 1 8 4 , 1093-1096 (1 974a). CASTRO,A. E., STEIN,N., HAKALA, T. R., and FRALEY, E. E., Characterization of a cell line from NELSON-REES, W. A., MCALLISTER,R. M., and human transitional cell cancer of the urinary GARDNER, M. B., Clonal aspects of the C-type virus-releasing cells of a cultured human rhabtract. J. nut. Cancer Inst., 53, 1341-1349 (1974). domyosarcoma line (RDI 14) in v i m . Nature New FIALKOW, P. J., MARTIN,G. M., KLEIN,G., CLIFBiology, 236, 147-149 (1972). FORD, P., and SINGH,s., Evidence for a clonal W. A., ZHDANOV, V. M., HAWorigin of head and neck tumors. Int. J. Cancer, NELSON-REES, THORNE, P. K., and FLANDERMEYER, 9, 133-142 (1972). R. R. HeLalike marker chromosomes and Type-A variant FOGH,J., and TREMPE, G., New human tumor lines. glucose-6-phosphate-dehydrogenase isoenzyme in In: Fogh (ed.) Human tumor cells in vitro. p. I 1 5human cell cultures producing Mason-Pfizer 159, Plenum Press, New York (1975). monkey virus. J. nut. Cancer Inst., 53, 751-757 GARTLER, S. M., Genetic markers as tracers in cell (1 9746). culture. Nut. Cancer Inst. Monograph, 26, 167-195 PETERSON, JR., W. D., STULBERG, C. S., and (1966). SIMPSON, W. F., A permanent heteroploid human KIRKMAN,H. N., Glucose-6-phosphate dehydrocell line with type B glucose-6-~hos~hate dehvgenase. I n : H. Harris and K. Hirschorn (ed.), droqenase. Proc. S o c . e x p . B i d . ( N . Y . ) , 135, Advances in human generics, Vol. 2, p. 1-60, Plenum 1187-1191 (1971). Press, New York (1971). PETERSON, JR., W. D., STULBERG, C. S., SWANLASFARGUES, E. Y., and OZELLO,L., Cultivation of BORG,N. K., and ROBINSON, A. R., Glucose-6human breast carcinomas. J. nut. Cancer Inst., phosphate dehydrogenase isoenzymes in human 21, 1131-1147 (1958). cell cultures determined by sucrose-agar gel and cellulose acetate zymograms. Proc. Soc. exp. LEVAN,A., Chromosome patterns in tumors. I n : Biol. ( N . Y.),128, 772-778 (1968). T. Caspersson and L. Zech (eds.), Chromosome identification, technique and applications in biology RASHEED, S., NELSON-REES, W. A., TOTH, E. M., and medicine. Nobel Symposium 23; Medicine and ARNSTEIN,P., and GARDNER,M. B., CharacNatural Sciences, Academic Press, New York terization of a newly derived human sarcoma cell (1973). line (HT-1080). Cancer, 33, 1027-1033 (1974). REED,M. V., and GEY,G. O., Cultivation of normal LIN, C. C., and GOLDSTEIN, S., Analysis of Qand malignant lung tissue. I. The establishment banding in human cell lines, J. nut. Cancer Inst., of three adenocarcinoma cell strains. Lab. Invesi., 53, 298-304 (1 974). 11, 638-653 (1962). MCALLISTER, R. M., GARDNER, M. B., GREENE, A. E., RHIM, J. S., CHO, H. Y., and HUEBNER, R. J., NonBRADT,C., NICHOLS, W. W., and LANDING B. H., producer human cells induced by murine sarcoma Cultivation in vitro of cells derived from a human virus. Int. J. Cuncer, 15, 23-29, (1975). osteosarcorna. Cancer, 27, 397-402 (1971).
81
NELSON-REES ET AL.
RHIM,J. S., CHO, H. Y., KIM, E. B., VERNON,H. L., NOLAN,J. F., and BYATT,P., Some properties of a ARNSTEIN,P., HUEBNER, R. J., GILDEN,R. V., new epithelial cell line of human origin. J. naf. Cancer Insf., 45, 107-122 (1970). and NELSON-REES,W. A., Characterization of non-producer human cells induced by Kirsten TOMPKINS, W. A. F., WATRACK,A. M., SCHMALE, mouse sarcoma virus. (MS submitted). J. D., SCHULTA, R. M., and HARRIS, J. A., Cultural SHIBLEY, G . P., HODGE, H. M., BANDYOPADHYAY, and antigenic properties of newly established cell M. F., ALLAN,L. J., CHAN,E., A. K., DEMAIO, strains derived from adenocarcinomas of human and O'CONNOR, T. E., I U D R induction and colon and rectum. J. nat. Cancer Inst., 53, 1101release of particles containing 70s R N A and 1110 (1974). reverse transcriptase from continuous-spinner cul- YOUNG,R. K., CAILLEAU, R. M., MACKAY,B., and tures of human liposarcoma cells. Proc. XIth InterREEVES,W. J., Establishment of epithelial cell national Cancer Congress, Florence, Italy, line MDA-MB-I 57 from metastatic pleural effusion October 20-26, 1974. of human breast carcinoma. I n Vitro, 9, 239-245 SINHA,A. K., and PATHAK,S., Distribution of (1974). constitutive heterochromatin in HeLa and Hep-2 ZALTA,A., MARUYAMA, K., DMOTHOWSKI, L., and cell lines. Humangenetik, 18,47-54 (I 973). BULTMAN,H., Karyological studies of transformed SOULE,H., VASQUEZ,J., LONG, A. S., ALBERT,S., human prostatic cell line (MA-160) by differential and BRENNAN, M., A human cell line from pleural banding. Presented at Southwest Section of the effusion derived from a breast carcinoma. J. nat. American Association for Cancer Research Annual Cancer Inst., 51, 1409-1416 (1974). Meeting, New Orleans, Louisiana, November 8-9, 1974. SYKES,J. A., WHITESCARVER, J., JERNSTROM, P.,
82
DISTINCTIVE BANDED MARKER CHROMOSOMES OF HUMAN TUMOR CELL LINES
Walter A. NELSON-REES, Robert R. FLANDERMEYER and Paula K. HAWTHORNE Cell Culture Laboratory, University of California, School of Public Health, Naval Biomedical Research Laboratory, Oakland, California, 9462S, USA
Nine human tumor cell lines (five breast carcinomas and four sarcomas) have been studied and each revealed groups of distinctive banded marker chromosomes which can serve to identify them and aid in monitoring cell line specificity. This was possible neither by conventional karyology in terms of numbers and morphology of chromosomes nor by glucose-6-phosphate-dehydrogenasemobility which was type B for all cultures. The SigniJcance of the clonal nature of the cell lines is discussed.
We recently detected HeLa-cell contamination of human cell cultures being utilized in several laboratories in this country and abroad (NelsonRees et al., 1974a, b). Our results on two cell lines, MA160 and H. Ep. -2, were confirmed by similar methods (Zalta et al., 1974; Lin and Goldstein, 1974). Our conclusions were based primarily on combined results obtained by two different methods with which previous workers had revealed HeLa cell-like characteristics, namely type A (fast) isoenzyme mobility pattern for glucose-6-phosphate dehydrogenase, G6PD (Gartler, 1966) and multiple banded marker chromosomes (Miller et al., 1971). The frequency in the general population of individuals with G6PD type A is relatively low (see Kirkman, 1971, for review) and one expects that only a fraction thereof develop neoplasms. Biopsies of only a few tumors are placed in culture and not all cultures grow up. It follows that the majority of cell lines in existence are of the type B (slow) mobility G6PD. Of these, many have achieved remarkable growth potential and, like HeLa, are widely used in many laboratories. Potential for contamination among such human, non-HeLa cultures therefore also exists, but we believe could be readily detected. We Received: March 10, 1975.
74
have selected a number of tumor cell lines exhibiting G6PD type B and have studied their chromosomes after Q and G banding. Unique groups of abnormal chromosomes (" markers ") were found in each cell line which can serve to distinguish them from each other and aid in monitoring their specificity. MATERIAL A N D METHODS
Cell lines Table I lists for each cell line studied the type of tumor of origin, the designation of the line derived from it, our immediate source and a published reference for each line. Cell culture method and chromosome analysis
All cells were grown as previously described (Nelson-Rees et a/., 19746). Methods for chromosome analysis were detailed previously (Nelson-Rees et a[., 1974a, b). Isoenzyme analysis The isoenzyme mobility pattern for G6PD was determined by Dr. W. D. Peterson, Jr., following his methods (Peterson et al., 1968).
4
VI
46-59
0
See addendum.
6
As human tumor cell culture re-isolated from cat (Nelson-Rees et al., 1972).
As recloned clone.
(Rasheed et al., 1974)
Fibrosarcoma HTlO80
S . Rasheed
0
0
late passage
* As clonal cultures.
a
11-27
?
9-1 7
62
33
3-26 '
18-39
124-128
Y
0
8-14
Y
6-10
18-31
P
0
24-42
Y
0"
T. O'Connor and G. Shibley (Chan et a / . , 1974)
R. McAllister (McAllister el a/., 1972)
R. McAllister (McAllister et al., 1971) J. Rhirn (Rhim et al., 1975)
E. Lasfargues (Lasfargues and Ozello, 1958) E. Lasfargues (Reed and Gey, 1962) M. Brennan (Soule et al., 1974) C. McGrath (McGrath ef al., 1974a) R. Cailleau (Young et al., 1974) G. Lowell (Fogh and Trernpe, 1975)
early passage
Liposarcoma SA, (strain B)
Rhabdomyosarcoma RDll4
R970 (derivative of Te85 subline F)
Osteosarcoma Te85 subline F
SKBR-3
MCF-7 (derivative of 734B) MDA-MBl57
734B
ALAB 496
Mammary carcinoma BT-20
TABLE I
44-48
59-65
51-67
43-52
51-53
50-59
46
64
66
46, 48
51
54
83
62, 64
51-68 75-102
87
74
54
49
79-89
68-76
53-57
43-53
TUMOR CELL LINES WITH G6PD TYPE B A N D DISTINCT MARKER CHROMOSOME PATTERN
+
+ translocated + translocated
-
-
-
-
-
-
-
-
-
v)
5
c
r
Gr
B
2
9
q
E
2
=!
0
3
U
t;
NELSON-REES ET AL.
RESULTS
Conventional staining methods demonstrated previously that morphologically aberrant chromosomes existed in these cell lines, but banding delineated and specified with greater precision each marker chromosome. Thus, for example, RDI 14 (Nelson-Rees et a/., 1972) had earlier revealed morphologically aberrant chromosomes including a ring, and HT1080, pseudodiploidy with aberrant chromosomes (Rasheed et a/.. I974), but banding demonstrated the specific origin of the “ marker ” chromosomes through rearrangement of normal chromosome banding patterns (Fig. I ) . For the present study, we first established the range and the modal number ofchromosomes in at least 20 metaphases of each cell culture (Table I). These figures alone could not serve to distinguish clearly between the different cell lines.
Q banding in at least 20 metaphases per culture revealed, as expected, absence of a Y chromosome in female cells and its presence in male-derived cells (Table 1). As previously noted (Nelson-Rees rt a/., 1974a), SA4 (strain B) cells exhibited an autosome-Y translocation. This chromosome is now designated as “ marker ” number 4 (M4) of this cell line (Fig. I). We studied at least 15 metaphases and constructed at least five karyotypes per cell culture following trypsin-Giemsa banding. All cell lines revealed groups of marker chromosomes as demonstrated by rearrangement of normal, human banding configurations. We concentrated o n all markers, up to a maximum of five, which were seen in all metaphases of individual cell lines at different passage levels (Fig. I ) . The five breast carcinoma cell lines exhibited the longest marker chromosomes observed. Although they were each clearly derived from different chromosomes (Table 11) the production of these long chromosomes most often involved most or all of the long arm of either chromosomes No. I , 2, or 3, the longest chromosomes in the normal karyotype. In two instances, cultures of the same line, but in use in different laboratories and passage levels, were studied and found to have identical markers. First, a culture of 734B was received at passage 3, grown and studied in our laboratory in 1972. The present marker chromosomes were from that culture.
76
Cultures of these cells were the subject of the work of Soule et a / . (1974). From the latter was derived a strain designated MCF-7 which is currently the subject of extensive research in viral oncology (McGrath c’f a/., 1974~7,b) l . MCF-7 was studied at passage 124 and banded in our laboratory in October, 1974. Both 734B and MCF-7 have a complex of identical marker chromosomes. Second, MDA-MB-157 cells at passage 6 were received and studied in our laboratory in May, 1973, prior to being described in the original publication (Young C? a/., 1974). Several markers studied in our culture are obviously identical to those appearing, but not described, in passage 3 cells i n that publication (6t.g. our No. I marker is indicated in the publication by an arrow). Among the sarcoma-derived cells another dual example is described. The osteogenic sarcomaderived Te85 cell line (McAllister et a/., 1971) was initiated and sublines established in 1969. The chromosomes of subline F shown are from passage 12. A derivative of this subline was recent I y u t i I ized in t ran sfornia t ion experiments with the Kirsten strain of murine sarcoma virus (Rhim et a/., 1975). The cells of the latter culture, designated R970, I8 passages after transformation, have markers identical to those of Te85 subline F. The same markers were observed (data not shown) in R970 cells when recovered following inoculation into *‘ nude ” mice where they induced tumors (Rhim et a/., MS submitted). SA4 (strain B) has been studied over many passages (Chan rt a/., 1974; Shibley et al., 1974; Bandyopadhyay et a/., 1974). In addition to the Y-autosome translocation mentioned above, we report a complex of marker chromosomes seen first at “ early ” and then at “ late ” passages following several clonings of the culture. However, one large submetacentric marker (M5) possibly not present in dl uncloned cells, was excluded from the culture in the course of cloning, but a complex of markers, including the Y-autosome translocation, persists. Cells of HT1080 received in 1972 at passage 8 and initially described by us as being pseudo-
* Drs. C. McGrath and M. Rich announced at the Xlth International Cancer Congress at Florence Congress that thecell line would be designated MCF-7 and the virus isolated from i t . 734.
FIGURE 1 Marker chromosomes observed in all cells of each cell line or its derivative. The proposed origins of banding patterns through rearrangements of normal human chromosomes are described in Table 11. Note: Cells of line SA4 (strain B) do not have a normal Y chromosome. Therefore, marker, number 4 of -‘early ” and ‘‘ late ” passages is described with the aid of a Y chromosome taken from a normal Y karyotype.
00
4
late *'
'
No. 1 1 with addition on q of portion of No. 3 q.
No. 5 with addition on p
of portion of No. 17 q.
7
Same
Same
No. 1 deletion of portion of p near centromere.
Same No. 9 with addition on q of portion of No. 8 q.
No. 16 with addition on p No. 17 with addition on p Small ring (on description). of portion of No. 14 q. No. 3 with addition on No. 21 with addition on p Y with addition on q of p of portions of No. 9 q portion of No. 15 q. of portion of No. 1 1 q. and No. 13 q. No. 9 with addition Same Same on q of portion of No. 13 q. of portion of No. 10 q.
No. 9 with deletion of p and addition on q of portion of No. 8 q,
No. 19 with addition on q of portion of No. 1Oq. No. 21 with addition on p of portion of No. 8 p.
No. 19 with addition on q of portion of No. 6 p.
No. 2 with addition on q of portion of No. 17 q (or X P).
M5
long arm of normal chromosome (Chicago Conference, 1966). Resembles HeLa No. 4 marker (see Nelson-Rees el a/.,19740) difference being that HeLa No. 4 has portions of weakly staining No. 22 and heavy bands of NO. I2 q
**
Same No. 5 with addition on q of portion No. 3 p. lsochromosome of No. I O q .
p = short arm; q
HT1080
*.
SAI *' early
Te8 5 (subline F) R970 RDI 14
of portions of No. 8 q and 2 q.
Same
No. 13 with addition on p No. 5 with addition on q of portion of No. 1 q. of portion of No. I q.
Same
No. 12 with addition on p No. 8 with addition on q of portions of No. 18 q and No. 2 q. No. 1 I with addition on q No. 7 with addition on q of portion of No. 5 q. of portion of No. 7 q.
Isochromosome of No. 2 q.
No. I 1 with deletion of portion of p.
of portion of No. 2 p.
No. 8 with addition on q
M4
No. 20 with addition on q No. 7 with addition on p of portion of No. 4 q. of portion of No. 10 q.
No. 14 with addition on p of portions of No. 18 q and No. 2 q. No. 12 with addition on q of portion of No. 12 q.
M3
SKBR-3
No. 3 p and centrornere with portion of No. 6 q.
No. 20 with addition of portions of No. 4 q and No. 12q. Same
No. 9 with addition on p of No. 4 q and most of No. 15 q.
No. 1 with addition on p of portion of X p.
M2
No. 2 with addition on q of portions of 5 q and 21 q.
Most of No. 1 1 with portions of No. 12 q and No. 5 q. No. 1 with addition on p of portion of No. 15 q (or X P). No. 20 with addition of portions of No. I 5 q and No. 1 q. Same
MI
MDA-MBI57
MCF-7
734B
ALAB
BT-20
Designation
DESCRIPTION OF POSSIBLE ORIGIN OF MARKER CHROMOSOMES IN HUMAN TUMOR CELL LINES
TABLE 11
DISTINCTIVE CHROMOSOMES OF TUMOR CELL LINES
diploid (Rasheed et al., 1974) were subsequently banded by Dr. W. F. Benedict at low and higher passage levels. Both passage levels carry markers presented here. DISCUSSION
Gartler’s hypothesis (Gartler, 1966) that many long-term human cell lines were, in fact, HeLa cells because, like HeLa, they exhibited the relatively rare type A mobility for G6PD, raised the question of whether prolonged cultivation of any human cell might not ultimately produce type A mobility (“convergence ”1. Some workers (Peterson et al., 1971) subsequently showed, however, that this was not necessarily the case. At least one cell line, Det 562, retained type B (slow) mobility throughout all passages in cultivation, ranging from 8 to 91. Our recent publications on cellular contamination (Nelson-Rees et al., 1974a, b) have again raised a similar question, namely whether all cell lines in cultivation for longer periods of time would not all acquire the same chromosome banding patterns as those demonstrated for HeLa and related cultures. We believe that the present results indicate that many cell lines, some now in long-term culture, retain their G6PD mobility and are unique entities, marked by peculiar and often complex chromosome banding patterns unrelated to those characteristic for HeLa cells. Undoubtedly, chance rearrangement alone could bestow upon any cell a “ marker ” chromosome similar or identical to one in another cell line, but the chances are minimal that entire complexes of multiple chromosome rearrangements as well as immunological characteristics would ultimately “ converge ”. Earlier data, now viewed in retrospect, conform to this concept. The first published description of a long-term cultivated human cell line with a unique banded chromosome marker and not considered to be of HeLa origin was, we believe, that by Miller et al. (1971) of WI-L2. This was a presumptive diploid lymphoblastoid line Dr. Benedict originally karyotyped the HT1080 cells by banding techniques and kindly informed us of his results (1973). We subsequently studied earlier passage material (see addendum in Rasheed el a/.,1974).
which was found to be pseudodiploid and carrying a Y-chromosome. Although we have not done trypsin-Giemsa banding on cells with that designation, a clonal derivative thereof also obtained from Dr. R. A. Lerner was studied by us several years later (Arnstein ei al., 1974) and found to show type B mobility for G6PD, also a Y-chromosome and still a modal number of 46 chromosomes. Similarly, Sykes’ ME180 cells of human cervical carcinoma (Sykes et al., 1970) as depicted, but not described, by Bordelon and Stubblefield (1974) showed no HeLa-like markers; our results (Arnstein et al., 1974) on cells with that designation received directly from Dr. J. A. Sykes indicated G6DP type B and a similar range in chromosome numbers. Elliot et al. (1974) described some G and C banded rearrangements in a line originating from a transitional cell carcinoma of the urinary tract. While the type of G6PD mobility is not indicated and the particular rearrangements are not defined, markers presented are, nevertheless, unique and may continue to serve to identify this cell line and its derivatives. One human report also utilizes banding patterns of marker chromosomes to distinguish a “ cell line ” from HeLa cefIs which it has been suspected of being for many years. H.Ep.-2 cell cultures from different sources have been indicted as HeLa cell contaminants (Gartler, 1966; Lin and Goldstein, 1974; Nelson-Rees et al., 19746). The work of Sinha and Pathak (1973) raises some doubt because it is not absolutely certain whether or not their results can be interpreted as describing two distinct strains of HeLa o r H.Ep-2 and HeLa as separate entities. It is possible that the small number of cells (4.5%) in the H.Ep.-2 culture which bore a Y chromosome were truly H.Ep.-2, but alternatively they may have been non-H.Ep.-2, non-HeLa contaminants. Of these, some might have also lost their Y. Nevertheless, those cells studied which lacked the Y “ h a d many features in common with the HeLa cells, including the st marker chromosomes ”. It would appear that additional immunological and enzyme data could serve to distinguish further between these sub-populations of the purported H.Ep.-2 cell culture and the HeLa culture, in order to distinguish between ‘‘ similarities ” and dissimilarities ” which in turn could either conform to or disagree with ‘‘ convergence. ” ‘I
79
NELSON-REES ET AL.
That contamination could be mistaken for ” or even for very convincing similarity between isolates of two separate tumors from different individuals was recently suggested by the use of banding techniques. We studied (Nelson-Rees, unpublished observations, 1975) two cell lines recently derived from adenocarcinomas of human colon and rectum (Tompkins et al., 19741, and sent to us by the originator. Both cell lines were G6PD type B and their cells carried a Y chromosome and showed aneuploidy involving the same chromosomes, as originally proposed by conventional staining. Neither, however, exhibited banded marker chromosomes (indicating re-arrangements) which could serve to distinguish these lines from each other. Pending results of further tests to define their identity, we presume as the originator now does (Tompkins, personal communication, 1975) that the two cell lines are one and the same. Clonal origin has been described for some, but not all, human tumors (see Kirkman, 1971 for review and Fialkow et al., 1972). It is tempting to ascribe multiple chromosome marker patterns specific for each cell line to unique events leading to formation of the individual tumors from which the cell lines were derived. Although our results conform to this concept, the distinct marker patterns may alternatively reflect “ stemcell line” (Levan, 1973) evolution and strict selection known to occur early in both tumorderived cultures and among long-term cultivated normal cells. Nevertheless, we have no direct chromosome data from the tumor specimens from which these lines were derived.
’‘ convergence
Like clones of HeLa cells or strains thereof maintained in separate laboratories, the cells studied here and other newer lines will acquire new markers through further rearrangements, or lose an occasional marker, naturally or due to cloning. However consistent monitoring and use of these markers as indicators will, we believe, permit identification of the cell lines and avoid cell contamination by newly established cell lines. AI>DENIlUM
After submitting this manuscript, we studied one additional culture from each of two lines at different passage levels and from other laboratories than those previously referred to in the body of the manuscript. BT-20 cells received from Dr. C. D. Aldrich at passage 274 had G6DP type B and clearly revealed marker chromosomes described here. SKBR-3 cells obtained from Dr. J. Fogh at passage 24 were also of type B G6PD mobility and at once revealed markers defined here. These results further confirm the value of banded marker chromosomes as persistent features of long-term cultivated cells. ACKNOWLEDGEMENT
We thank Dr. W. D. Peterson, Jr. for the isoenzyme mobility tests and Mr. I . P. Madin for assistance in karyology. This work was supported by Contract E 73-2001-NOl-CP-3-3237 in the Virus Cancer Program, National Cancer Institute, National Institutes of Health.
CHROMOSOMES MARQUEURS DlSTlNCTlFS DANS LES LIGNEES DE CELLULES TUMORALES HUMAINES Neuf ligiiL;rs de cellules tunlorales humaities ( 5 cancers du seiri ef 4 sarcotnes) otit Lte c5trrdiees; chacune contenait des groupes de chrotiiosonies marqueurs distinctifs que l’on a rep6rc;s en utilisant la technique des barides et qui peuvent servir a identifier les lignles cellulaires et aider contrder leur spkcificiti, ce qiri n’est pas possible par la caryologie classique, c’est-a-dire d’aprks le tiombre et la niorphologie rles chromosomes, ni d’aprks la tnobiliti de la glucose-6-phosphate-d~shydrogina.se, yiri etait de type B pour toutes les euftures. Les auteurs atialysent I’itnportunce cle la nature clorialc cles lignivs cellirlaires.
80
DISTINCTIVE CHROMOSOMES OF TUMOR CELL LINES
REFERENCES
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NELSON-REES ET AL.
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82
