Chromosome Studies of 8 in Vitro Lines of Burkitt's Lymphoma1

(CANCER RESEARCH 27 Part 1, 392-402,February 1967] Chromosome Studies of 8 in Vitro Lines of Burkitt's Lymphoma1

CHARLES P. MILES2 AND FRANK O'NEILL2

Division of Pathology, Sloan-Kettering Institute for Cancer Research, and Memorial Hospital, New York, New York

Summary Chagrin Falls, Ohio) supplemented with 2.2 gin/liter of NaHCO and 12% fetal calf serum. The cells are grown in 50-ml medicine Of 8 Burkitt lymphoma lines, 4 had classically diploid stem- bottles with about 10 ml of medium. Cells do not attach to glass lines, Ogun and Kudi being female, and Raji and EB3, male. but do settle to the bottom so that medium can be removed by EB2 was diploid but with an abnormal near-metacentric marker. decanting. Medium is changed 3 times weekly. When the cell SLi was diploid but with 3 markers. EB1 was subdiploid. Jiyoye suspension appears too concentrated, the bottle is agitated and was normal male except for trisomy for a 6-12 -fX chromosome. half the suspension passed into a 2nd bottle. As compared with benign cells, the lines tended to show a high Chromosome analysis is done 24 hr after change of medium. incidence of aberration including markers, multiradials, and One drop of 0.01% colchicine is added and the bottle then pulverized metaphase figures. A sporadic marker, RM-1, has incubated for another 75-90 min. Chromosome preparations are been observed in SLi, Jiyoye, and Ogun. Distinctive secondary made essentially according to the method of Moorhead et al. constrictions occur on a pair of 6-12 +X homologs in EB2, EB3, (12, 16). and Jiyoye. Metaphase figures which ap[)eared to be unbroken were photographed and numerous karyotypes were made for each line. Introduction Since some of the initial photographs were taken because the EB1 (4), EB2 (6), EB3 (5), SLt (22), Jiyoye, Ogun, Kudi, and cells exhibited unusual markers or other abnormalities, a further Raji (19, 20) are permanent tissue culture strains of lymphoid microscopic random study was done in which 200 consecutive cells derived from lymphosarcomas (Burkitt's lymphoma) in analyzable metaphase figures were scored for the presence of African children. In addition to their oncologie interest, these unequivocal markers (such as abnormally long acrocentrics, human cell lines are most unusual in that they maintain lymphoid minutes, rings, dicentrics, fragments, etc.); multiradials; frag morphology, grow in suspension culture, and are all diploid or mented or pulverized metaphase figures; and cells having near-diploid. The latter 2 characteristics may make the lines approximately twice the stemline number (Table 1). In the case useful experimental tools. Counts and stemline karyotypes have of EB2, 50 consecutive diploid (or diploid plus 1 or 2 chromo been reported for SLi and EB2 (22). From the description, Sl^ somes) cells were karyotyped as well in an effort to determine the has a karyotype very similar to that in ALI, a cell strain isolated true incidence of variation from the stemline karyotype (since the from the same tumor by Rabson et al. (21). More recently, Cooper scoring by simple inspection of 200 cells would miss those eÃal. (1) have described stemline numbers and other features of karyotype variants where there were numerically balanced EB1, EB2, and EB3. The following report provides further gains and losses of nonmarker chromosomes). cytogenetie data on these and the other lines. The extent and kind of chromosome abnormality are described Results in some detail in order to show the differences from one cell line Ogun and Kudi, Raji anil EB3 all appeared to have classically to another and from benign human cell strains. Such data may diploid stemline karyotypes, the former 2 being female and the also shed light on the cellular mechanisms producing the ab latter 2 male. See Figs. 1-3, and 5. Jiyoye had a 47 chromosome normalities as well as enabling comparison with changes induced stemline, normal male except for an extra 6-12 +X chromosome experimentally by drugs, viruses, or other agents. (Fig. 4). EB2 was diploid but the stemline karyotype contained an abnormal slightly submedian chromosome intermediate in Materials and Methods size between a Xo. 3 and a Xo. 6 (Fig. 6). (However, a substrain Technics for growing lymphoma lines have been described by of EB2 recently recovered from frozen storage had stemlines at Epstein (4), Stewart et al. (22), and Rabson et al. (21). We have 47 and 48.) cultured these cells in Eagles basal medium with L-glutamine and Both EB2, EB3, and occasionally Jiyoye exhibited a dis Earles salts (supplied in ]x>wderedform by General Biochemicals, tinctive near-terminal secondary constriction in the long arm of 1 or 2 6-12+X chromosomes of similar size (i.e., where the 1This work was supported by Grant DRG 798 from the Damon constriction appears on 2 chromosomes, the affected chromosomes appear to be homologs) (Figs. 5-7). This constriction appeared Runyon Memorial Fund for Cancer Research and by NCI in about 5-10% of EB2, 2-3% of EB3, and less than 1% of CA08748. 'Present address: Pathology Department, University of Utah Jiyoye cells. We have also observed it in occasional cells of a Medical Center, Salt Lake City, Utah. strain of leukemia, LK63 (recently isolated by Dr. Donald Received June 24, 1966; accepted September 28, 1966. Armstrong of the Sloan-Kettering Institute). Both Jiyoye and

392 CANCER RESEARCH VOL. 27

TABLE 1 DISTRIBUTIONOFVARIANTSIN200Â°CONSECUTIVECELLSFROMEACHLINEANDFROMWI38CELLSWITH:

Sordiplo- Xo. with chromo- S with S with than tation S with multi- NameEBlÂ«EB2EB3SLiJiyoyeOgunKudiRajno markers615156404710Multi- somes markers23106020002 fragmen numerical obviousab No.Â±23530343325303129Obviousradials010102000Fragmen(pulveriza withoutother radials0000020002tation010104000Variants(otherbut in tion)10217060002ab cluding2 normality114153102163109143147151Stemline normality6073803797112 S)Wo)162578028845

iWI38"Exactstendine

0 A few cells with Â±3ormore chromosomes are omitted from the table. 6Obvious markers here include very small or very large elements, rings, dicentrics, and fragments (other than those appearing in the stemline karyotype). cSince the exact stemline number is uncertain for EB1, variants from it are not tabulated. d WI38, a fibroblast strain used for comparison, was isolated by Hayflick (6). Our substrain was ob tained from Flow Laboratories, Rockville, Maryland.

TABLE 2 HOMOLOGDISTRIBUTIONIN23 KARYOTYPES

GROUP1 TYPEOK No.OFSUCH OF CHROMO CELLS2231111111111111111Xo.SOMES46464746464646464646464646464747474545CHROUOSOUE (2)Â«22122222212222222222(2)1111111,1111111111113(2)22233223333222232124-5(4)42434222152434353536-12+Xacrecen trie submedian marker11 (15)1315141313151514141213131413161215121313-15(6)656566656556466665616-18(6)67675767678776677761Â»-20(4)444434344443433334321-22(4)4444444454445444444Y(D1111111111111111111Longmarker1111111111111111101Longmarker2222212222222222212Other

(dicentric)11

(dicentric)2

(1 a dicen tric)1 (dicentric) 1Numbers in parentheses indicate number expected for normal cells.

Ogun showed occasional prominent constrictions at other sites variation is illustrated in Table 2 which shows these distributions but usually only on a single homolog. A constriction on the short for 23 karyotyped cells. These karyotypes were made without arm of what is probably Chromosome No. 7 or 8 was very common comparing one with the other and only later were attempts made in Ogun cells (Fig. 1). EB1 and SLi exhibited a very high inci to reconcile differences (e.g., by reassigning ambiguous chromo dence of karyotype variation. In the case of SLi, although most somes). However, even "forced" attempts at reconciliation did of the cells showed the same 3 distinctive markers, they usually not significantly decrease the high incidence of variation. Only 3 differed in the distribution of various homologs. This marked (Figs. 9, 10), 2 and 2 (Fig. 8) cells, respectively, had the same

FEBRUARY 1967 393

TABLE 3 HOMOLOGDISTRIBUTIONIN28 EB1 KABYOTYPES

GROUP1 TYPEOR No. OF SUCH OFCHROMOSOMES45444545454545454545454545454545444444444444CHROMOSOMF. KARYOTYPES23111111111111111No. 2+X (2)"22222222222223222221222(2)22112212222213112221113(2)33433333222231222333344-5(4)44444444544444445444446-1(16)1515161015141615151614151615161614121317141713-15(6)565566645666655666565516-18(Â«)655656655665656656645519-20(4)444444454444444444444321-22(4)3333333443443434334343Markers111111111212112

0 Numbers in parentheses indicate number expected for normal cells. karyotype. One of these 3 karyotypes was quite atypical since it TABLE 4 had a long submetacentric marker in lieu of a No. l homolog, and INITIALANALYSES this marker was present in only 4 of the karyotyped cells. What appears to be the sanie long submetacentric marker has also age of line of NameEB1EB2EB3SL,JiyoyeOgunKudiRajsomalsex?XXXYXYXYXXXXXYStemlincpioid?NoNoYesNoNoYesYesYesMinimum(mo.) asofJan. karyotypes2930422320401410Variantkaryotypes appeared sporadically in Jiyoye (Fig. 11) and in Ogun (Fig. 12). No.44-4546464647464646Classicallydi1966"2520121521212854No.(Â°^Y\/Qf7960219110451410 It also occurs in all analyzed cells of a primary malignant lymphoma, and of a metastatic breast cancer, as well as in a solitary karyotype variant in another primary lymphoma (10). In each instance it is (a) the largest element in the karyotype, (6) it re places a No. 1, and (c) it is distinctly more submedian than is Chromosome No. 1. We therefore suggest that this is a marker chromosome which occurs in more than 1 tumor and we projxise provisionally to label it RM-1 for Recurrent Marker, No. 1. iChromo Alternatively, it might be called the Madison chromosome, since it is conceivably identical with the augmented No. 1 originally â€¢Someof these lines may be older but the precise date of origin described by Patau et al. (17) in Madison, Wisconsin. In con is not always specified in the original reports. trast the marker in the Ogun karyotypes of Figs. 13 and 14 is not a recurrent marker in the sense implied above, since thus far we 3 chromosomes and only 3 short acrocentrics, and this was a have observed it only in the Ogun cell line, and the examples in common abnormality of the stemline. Of the 27 cells karyo these 2 Ogun figures probably did not arise independently. typed, 17 had 3 No. 3's, and 19 had only 3 short acrocentrics. One of the other two SLi karyotypes with 46 chromosomes Thus while the karyotypes had common features, a dominant (Fig. 8) is almost identical with that pictured by Stewart et al. (22) stemline karyotype could not be defined except as a theoretical except for the presence of an additional long acrocentric marker entity. The only cell with 46 chromosomes was pseudo- in lieu of a 6-12 chromosome. All but 2 of the 23 karyotyped cells diploid (Fig. 19). exhibited the 3 distinctive markers. The incidence of variant karyotypes for the 8 strains is shown As seen in Table 3, of 27 EB1 karyotypes, all but 1 contained in Tables 1 and 4. Table 4 represents the first cells photographed 44 (9 cells) or 45 (17 cells ) chromosomes. Three of the karyotypes and in some instances these were selected because they were with 44 chromosomes were identical as were 2 with 45. See Figs. unusual. For Table 1, cells were examined at random. It may be 15 and 16, and 17 and 18. However, all 5 of these cells had 3 No. seen that rates of variation suggested by the selected karyotypes

394 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1967 American Association for Cancer Research. In Vitro Studies of Burkitfs Lymphoma in Table 4, is born out by the randomized analyses in Table 1. In Table 6 lists the homologs involved in variation from the general, those lines which showed a high percentage of variant stemline karyotype for all lines except KB1 and SLj (since for karyotypes in the metaphase figures initially photographed, also these latter 2 we cannot be sure as to the precise stemline karyo showed a high incidence of abnormalities, and conversely. type). It may be noted from this table that certain chromosome Various examples of sporadic, or recurrent markers; multi- groups, and particularly the F (19-20) group, tended to vary less radiais; secondary constrictions; fragmented and polyploid cells than do the others. are shown in Figs. 8-14 and 20-25. Surprisingly, Jiyoye, despite There was a strong tendency in these cell lines for several its 47 stemline, showed an incidence of variation lower than that abnormalities to occur in the same cell. This was particularly in a benign fibroblast strain, WI38. While neither of these culture true of tetraploid or near-tetraploid cells. In extreme cases many strains showed obvious abnormalities in 200 cells scored, WI38 chromosomes showed secondary constrictions or were affected by has an approximately 5% incidence of tetraploidy while Jiyoye breaks and exchanges. These cases tended to fall into rather showed no tetraploid (or, more precisely, 2S) cells. sharply defined categories without much admixture. In inverse Table 5 illustrates that when all varieties of abnormality (i.e., order of incidence these were: (a) multiple secondary constrictions including pseudodiploid cells without obvious markers) are involving many chromosomes (while constrictions in 1 or 2 considered, the variation rate was even higher. The selected chromosomes were common, constrictions in many, as in Figs. 20 karyotypes of Table 4 and the analysis of variant karyotypes and 21, were rather uncommon); (6) figures with multiple chroma- tabulated in Table 6 suggest that total variation was corre lid breaks and exchanges thus forming multiradials (Fig. 22); and spondingly high for lines other than EB2. (c) figures with multiple chromosome breaks and reunions to form dicentric or multicentric chromosomes (Fig. 23). More TABLE 5 marked fragmentation (pulverization) (Fig. 24) may be a more TYPESOFKARYOTYPEVARIANTSIN50 CONSECUTIVECELLS,EB2 extreme version of the same process. In the latter cells there was not infrequently a tendency for pulverization to be restricted Xo. of No. of to 1 area of the cell (Fig. 25). chromo Xature of karyotype such cells somes

-Iti Stemline karyotype Discussion 45 Includes a dicentric 2 It would appear from these date that the numerical stemlines 45 Includes a multiradial 2 46 Monosomy, 13-15, includes a long acrocentric 2 of permanent human tissue culture strains are in part a function marker of cell type. Recently isolated but apparently permanent cell lines 46 Includes a dicentric 1 of lymphoid cells have all been diploid or very near-diploid. Cell 46 Trisomy, No. 1, monosomy, No. 3 1 lines of epithelial origin are all near-triploid or have higher 4(1 Trisomy, 6-12 + X, monosomy, No. 2 1 numbers, although there is one exception, RI'MI 2650 (12-14). 47 Trisomy, No. 2 1 The chromosome analyses are somewhat similar to those 47 Trisomy, 6-12 + X 1 reported by Jacobs et al. (8) for a series of cases of Burkitt's 47 Includes a minute 2 lymphoma done on biopsies. The stemlines tended to be diploid 47 Trisomy, No. 1 and a 21-22, monosomy, No. 2 1 or near-diploid. This tends to be true of primary lymphosareomas Trisomy, 4-5 1 47 in general as op]>osed to the more pleomorphic reticulum cell 48 Includes a minute and a fragment 1 sarcomas which are more often markedly aneuploid (10, 11).

TABLE 6 LOSSESORGAINSOF PARTICULARHOMOLOGSINVARIANTSFROMTHESTEMLINEKARYOTYPES"

GROUPXo. No. OB NAHEEB2EB3OgunJiyoyeKudiTotalsAbsolutesumsNo.OFVARIANTS25592243CHROMOSOME 1+ 2+ 3+

200-10+2, 1,-100+ 1,-1+ 1,-4+ -7+ 10000+1121-22+10-200+ 1,-1-100+ 1-200+2, 1,-1+2, -1+2+ 10+2, 1+8,

-13No. -13No. 2, -354-5+ -686-12+X+2,-917li-15-40-200-6616-18-1-1-10+1+1, -3419-20+ 1, -23

" These data are skewed at least to the extent that cells with fewer than 46 centromeres were usually not karyotyped on the assumption that chromosomes had been artifactually lost. This assumption is probably not always true. EB1 and SLi are omitted, since it was not possible to establish unequivocal stemline karyotypes for these lines. Raji is omitted, since the only variant karyotype contained only 37 chromosomes.

FEBRUARY 1967 395

Thus while the possible benign origin of supposed cancer cell which do produce mature virus. Until the significance of virus strains is always a problem, the fact that these lines are at least (or virus-like) particles observed in the Burkitt lymphoma lines near-diploid is not inconsistent with their being cancer cells. has been elucidated, one must draw inferences with great caution. The significance of the peculiar secondary constrictions in Nevertheless it is tempting to second (albeit in a different con EB2, EB3, and Jiyoye is uncertain. We have observed distinctive text) the hyiwthesis favored by MacKinnon et al. (9) "that the secondary constrictions in a number of tumors but usually, as chromosomal aberrations develop and persist as a direct result of with the short arm constriction on Chromosome No. 7 or 8 in a viral genome." Ogun, in only one homolog and therefore similar in randomness to the other presumably random structural abnormalities so Acknowledgments common in cancer karyotypes. Since the secondary constriction in EB2, EB3, and Jiyoye so often occurs in what seem to be both We thank Mrs. Andrea Scheldt and Mrs. Margaret Johnson for homologs, it does not appear to result from a random event. their assistance. Similar constrictions on what may be the same homolog occur in RPMI 2650 but in association with a generalized enhancement of References secondary constrictions. \Vhether these constrictions have onco- genic significance is a question which cannot as yet be answered. 1. Cooper, E. H., Hughes, D. T., and Topping, N. E. Kinetics and Chromosome Analyses of Tissue Culture Lines Derived It may be of interest that the feature observed in many primary from Burkitt Lymphomata. Brit. J. Cancer, 20: 102-13, 1966. lymphomas, i.e., accentuated secondary constrictions in what is 2. Cooper, H. L., and Hernits, R. A Familial Chromosome Vari presumed to be Chromosome No. 9 (10, 11) have not usually ant in a Subject with Anomalous Sex Differentiation. Am. J. been seen in these tissue culture lines. The chromosome with Human Genet., 15: 465-75, 1963. near-terminal secondary constrictions never showed a constric 3. Davidson, E. H. Differentiation in Monolayer Tissue Culture tion near the centromere although these peculiar chromosomes Cells. Advan. Genet., le: 143-280, 1964. sometimes appeared in the same figure with presumed 9's. How 4. Epstein, M. A., and Barr, Y. M. Characteristics and Mode of ever, a feature shared both by the Burkitt lines and by primary Growth of a Tissue Culture Strain (EBI) of Human Lympho- blasts from Burkitt's Lymphoma. J. Nati. Cancer Inst., 34: lymphomas (11) is a tendency for Group F (19, 20) and to a lesser extent Groups E (16-18) and G (21, 22) to be spared by the 231-40, 1965. 5. Epstein, M. A., Barr, Y. M., and Achong, B. G. Studies with mechanisms which add or subtract homologs. Burkitt's Lymphoma. In: Symposium on Methodological Ap Despite some overlap there seem to be at least 2 varieties of proaches to the Study of Leukemias, Wistar Inst. Monograph extreme chromosome damage, one involving chromatid, the No. 4, pp. 69-82, 1965. other chromosome breaks. Perhaps these differences relate to the 6. . The Behaviour and Morphology of a Second Tissue stage of cell cycle at which the damage occurs. Culture Strain (EB2) of Lymphoblasts from Burkitt's Lymph As mentioned, the marker chromosome which we have labeled oma. Brit. J. Cancer, 19: 108-15, 1965. RM-1 may be identical with the augmented No. 1 originally 7. Hayflick, L., and Moorhead, P. S. The Serial Cultivation of described by Patau et al. (17). Human Diploid Cell Strains. Exptl. Cell Res., go: 585-621, An apparently identical chromosome has been described by 1961. 8. Jacobs, P. A., Tough, I. M., and Wright, D. H. Cytogenetic Yunis and Gorlin (23), Cooper and Hernits (2), and Philip et al. Studies in Burkitt's Lymphoma. Lancet, //: 1144-46, 1963. (18). Perhaps this anomaly can be produced by more than 1 agent 9. MacKinnon, E., Kalnins, V. L, Stich, H. F., and Yohn, D. S. affecting chromosomes. Comparative Karyologic and Immunofluorescent Studies on It has been previously argued that karyotype instability is a Syrian Hamster and Human Amnion Cells Infected with common feature in some cancers (12). This process is similar to Human Adenovirus Type 12. Cancer Res., 26: 612-18, 1966. that described by Davidson (3) for permanent tissue culture 10. Miles, C. P. Chromosome Studies on Solid Tumors. I. 28 Non- lines whether of benign or cancerous origin. Nevertheless, the epithelial Tumors. Cancer, in press. degree of instability varies markedly both in these Burkitt 11. Miles, C. P., Geller, W., and O'Neill, F. Chromosomes in lymphoma lines and probably in cancers in general (10). As Hodgkin's Disease and Other Malignant Lymphomas. Cancer, mentioned above, we have recovered from frozen storage a 19: 1103-16, 1966. substrain of EB2 which has stemlines at 47 and 48. This probably 12. Miles, C. P., and O'Neill, F., Chromosomes of Some Hetero- represents selection of variant cells. Possibly the EB2 subline of transplanted Human Tumors. II. H.Ep. #1, H.Ep. #3, and Cooper et al. (1) with a predominant chromosome range of 86-88 H.Ep. # 5. J. Nati. Cancer Inst., 35: 435-58, 1965. represents selection of near-tetraploid variants. As seen from 13. . Prominent Secondary Constrictions in a Pseudo- Tables 1, 4, and 5, EB2 has a high rate of variation. Also in diploid Human Cell Line. Cytogenetics, 5: 321-34, 1966. contrast with our findings are those of Cooper et al. with respect 14. Moore, G. E., and Sandberg, A. A. Studies of a Human Tumor to EB1. They report a diploid stemline as opposed to our findings Cell Line with a Diploid Karyotype. Cancer, 17: 170-75, 1964. of subdiploidy. 15. Moorhead, P. S. Human Tumor Cell Line with a Quasi-diploid Both the karyotype instability as well as some of the varieties Karyotype (RPMI 2650). Exptl. Cell Res., 39: 19-196, 1965. of metaphase damage are similar to effects produced experi 16. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, mentally with viruses. MacKinnon et al. (9) report that with D. M., and Hungerford, D. A. Chromosome Preparations of adenovirus Type 12, marked cytogenetic abnormalities occur in Leukocytes Cultured from Human Peripheral Blood. Ibid., hamster cells where mature virus is not produced, whereas 20: 613-16, 1960. aberrations are not increased in infected human amnion lines 17. Patau, K., Therman, E., Inhorn, S. L., Smith, D. W., and

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Ruess, A. L. Partial-trisomy Syndromes. II. An Insertion as 21. Rabson, A. S., O'Conor, G. T., Baron, S., Whang, J. J., and Cause of the OFI) Syndrome in Mother and Daughter. Chro- Legallais, F. Y. Morphologic, Cytogenetic and Virologie mosoma, 12: 573-84, 1961. Studies in Vitro of a Malignant Lymphoma from an African 18. Philip, J., Frydenberg, O., and Sele, V. Enlarged Chromosome Child. Intern. J. Cancer, /: 89-106, 1966. No. 1 in a Patient with Primary Amenorrhoea. Cytogenetics, 22. Stewart, S. E., Lovelace, E., Whang, J. J., and Ngu, V. A. 4: 329-39, 1965. Burkitt Tumor: Tissue Culture, Cytogenetic and Virus Stud 19. Pulvertaft, R. J. V. Cytology of Burkitt's Tumour (African ies. J. Nati. Cancer Inst., 34: 319-27, 1965. Lymphoma). Lancet, /.- 238-40, 1964. 23. Yunis, J. J., and Gorlin, R. J. Chromosomal Study in Patients 20. . A Study of Malignant Tumors in Nigeria by Short-term with Cysts of the Jaw, Multiple Nevoid Basal Cell Carcino- Tissue Culture. J. Clin. Pathol., 18: 261-73, 1965. mata and Bifid Rib Syndrome. Chromosoma, 14: 146-53, 1963.

FIG. 1. Stendine karyotype for Ogun. Note the short arm gap or secondary constriction on one of the 6-12+X homologs (here placed as No. 7). All the karyotypes are arranged in accord with the Denver conventions except in the placing of Groups 1 through 4-5 on the same line. Where marker chromosomes appear they are placed to the right of the 13-15 group. FIG. 2. Stemline karyotype for Kudi. FIG. 3. Stemline karyotype for Raji. The Y chromosome is frequently somewhat more distinctive than the one shown here. FIG. 4. Stemline karyotype for Jiyoye. 47 chromosomes. Y plus trisomy C. FIG. 5. Stemline karyotype for EB3. The Y (last chromosome in last row) is distinctive. Note the long arm secondary constrictions on two 6-12+X homologs (arrows). FIG. 6. Stemline karyotype for EB2. The 1st chromosome in the 2nd row (6-12+X group) is probably a marker. In some cells what appears to be the same marker is nearly as large as a No. 3. Note the near-terminal secondary constrictions on two 6-12+X homologs (arrows). FIG. 7. Further examples of 6-12+X chromosomes with near-terminal secondary constrictions (a and 6) EB3, (c) Jiyoye. FIG. 8. Stemline karyotype for SLi . Almost all of the cells karyotyped had the 3 marker chromosomes and lacked a No. 2. FIG. 9. SLi . This karyotype is identical with that in Fig. 10, and contains a long submedian marker (HM-1) in lieu of a No. 1 chro mosome (arrow). FIG. 10. SLi . Another karyotype with the HM-1 marker. FIG. 11. Jiyoye. A karyotype with the RM-1 marker. FIG. 12. Ogun. A karyotype with the RM-1 marker. FIG. 13. Ogun. A karyotype containing a satellited submedian marker which thus far has been observed only in Ogun cells. FIG. 14. Ogun. This karyotype contains a satellited submedian marker apparently the same as that in Fig. 13. In addition, there are 2 multiradial forms. FIGS. 15 AND16. EBl. Two apparently identical karyotypes; 44 chromosomes. As here, most karyotypes lacked a small acrocentric and had an extra Chromosome No. 3. FIGS. 17 AND18. EBl. Identical karyotypes containing 45 chromosomes including an abnormally long acrocentric. FIG. 19. EBl. A pseudodiploid karyotype; 46 chromosomes. Seven chromosomes have been placed in the 13-15 group but 1 or more of these is probably abnormal. FIG. 20. EB2; 47 chromosomes including at least 4 markers. Secondary constrictions on several chromosomes. On 2 chromosomes (arrows) these are near-terminal and resemble satellites. FIG. 21. SLi . Highly polvploid cell showing multiple secondary constrictions. FIG. 22. Ogun. Breaks with multiple chromatid exchanges (multiradial forms). FIG. 23. Ogun. Multiple chromosome breaks with reunions to form multicentric chromosomes. FIG. 24. EBl. Fragmentation (pulverization). Note that a portion of the periphery of the figure appears less affected. FIG. 25. EB2. More extreme fragmentation restricted to 1 area of the figure.

FEBRUARY 1967 397

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Charles P. Miles and Frank O'Neill

Cancer Res 1967;27:392-402.

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