translocation related to the Philadelphia chromosome. MITELMAN, F. 1974. Heterogeneity of Phl in chronic myeloid leukaemia. -~ Hereditas 76: 315---316.
304
B R I E F REPORTS
HAYATA, 1.. KAKATI, S. and SANDBERG, A. A. 1973. A new translocation related to the Philadelphia chromosome. Lance/ ( 2 ) : 1385. F. 1974. Heterogeneity of Phl in chronic MITELMAN, myeloid leukaemia. -~ Hereditas 76: 315---316. Department of Internal Medicine, University of MITELMAN, F., BRANDT,L. a n d NILSSON,P. G. 1974. Lund, Sweden Cytogenetic evidence for splenic origin of blastic transformation in chronic myeloid leukaemia. -. Scand. J . Haeniatal. 13: 87-92. Literature cited NOWELL,P. C. a n d H U N G E R F O RD. D , A . 1960. A minute chromosome in human chronic granulocytic leukemia. CASPERSSON. T., GAHKTON, G., LINDSTEN, J. and ZECH,L. Sc,irncr 132: 1497. 1970. Identification of the Philadelphia chromosome a s a ROWLEY,J. D. 1973. A new consistent chromosomal number 22 by quinacrine mustard fluorescence analysis. abnormality in chronic myelogenous leukaemia identi-- Exp. Cell RFS.6 3 : 238 -240. fied by quinacrine fluorescence a n d Giemsa staining. ENGEL.E., MCGEE,8 . J., FLEXNER, J. M . RUSSEL,M. T. Natirre 243: 290-293. a n d MYERS,B. J. 1974. Philadelphia chromosome (Phl) SANDBERG. A. A. and HOSSIELD,D. K . 1970. Chromotranslocation in a n apparently Phl negative, minus G22, somal abnormalities in human neoplasia. Ann. Rev. case of chronic myeloid leukemia. - Ne" EngI. J . Med. M d . 21: 3 7 9 ~408. 291: 154. P. P., W H A N GJ., a n d FREI,E. 1966. EZDINLI,E. Z., SOKAL.J. E., CROSSWHITE, L. and SAND- TJIO,J. H., CARBONE, The Philadelphia chromosome and chronic myelogenous BERG, A. A. 1970. Philadelphia chromosome-positive leukaemia. J. Nat. Cancrr Inst. 3 6 : 567 - 584. a n d -negative chronic myelocytic leukemia. Ann. WHANG-PENG. J . , CANELLOS. G . D., CARBONE, P. P. and Internal Med. 7 2 : 175 182. TJIO, J . H. 1968. Clinical implications of cytogenetic FOERSTER, W., MEDAU,H. J. a n d LOFFLER,H . 1974. variants in chronic myelocytic leukemia (CML). Chronische myeloische Leukamie mit Philadelphia BloocI32: 755 ~-766. Chromosom und Tandem-Translokation a m 2. Chromosom Nr. 22; 46, XX. tan (22q+ ; 2 2 q - ) . . Klin. Wochmsclir. 52: 123 126. Felix Mitelman GAHRTON,G., ZECH, L. a n d LINDSTEN, J. 1974. A new Department of Internal Medicine variant translocation ( l 9 q + , 22q - ) in chronic myeloUniversity Hospital cytic leukemia. E x p . Cell Res. 8 6 : 214 216. S-221 85 Lund, Sweden A ~ k n o ~ ~ l c ~ t l ~ t ~ r cThis ~ n t .work \. was supported by grants froni the Swedish Cancer Society and J. and A . Persson Foundation for Medical Research.
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~
~~
~~
~~~
~
JOACHIM MARK,JAN PONTENand BENGTWESTERMARK: Cytogenetical studies with C - b a n d technique of established cell lines of h u m a n malignant gliomas (Received September 13, 1974) Human turnours in the nervous system have been subjected to comprehensive cytogenetical studies during the last years. The results of these studies were recently surveyed (MARK1974). Among the various primary malignant tumour types, the astrocytic gliomas constituted the predominating group. As far as only conventional staining methods are considered, the chromosomal characteristics of these gliomas are now known in detail (MARK 1971). It has been possible, however, to carry these analyses further applying modern banding methods to established glioma cell lines. The observations with G-band technique in two such cell lines were previously discussed (MARK et al. 1974a, b), and the present communication deals with the cytogenetical findings in a third glioma cell line, 343 MG. Hereditas 78, I974
Material and methods The established glioma cell line 343 MG was derived from a temporal astrocytic glioma (grade 1Ii) in a 64-year-old male. The initiation and the maintenance of the cell line have been described earlier ( P O N T ~and N MACINTYRE 1968; WESTERM A R K et al. 1973). Cytogenetical analyses were performed on 343 M G after nearly three years of in vitro growth. Several preparations from passages 21 1-220 were used. Since the observations in different preparations agreed they are combined below. The methods for the chromosome preparations, including the G-banding technique as well as conventional staining methods, have been described previously (MARKet al. 1974 a, b).
BRIEF REPORTS
305
Table I. The distribution of chromosome counts in the glioma cell line, 343 MG ~
~~
~
Chromosome numbers
Total cells
6069
70 71
72
73
74 75
2
1
5
6
1 0 3 5 9
3
The chromosomes were counted in a total of 100 cells. Magnified photos were used for the karyotype analyses which comprised 35 cells. The nomenclature follows that of the Paris Conference (1971) with two exceptions: (1) S is used as a symbol for the stemline; (2) all structurally changed chromosomes are termed markers (mar) regardless of whether or not their origin is wholly or partly identified.
mar
mar Results
Table 1 shows the distribution of the chromosome counts. There was a peak at S = 75, aconsiderable spread around the modal number, and only a few cells with chromosome numbers in the double stemline region. Ten of the 17 karyotyped cells with the S-number had the same karyotype, the stemline karyotype. This is shown in Fig. 1 . The S-karyotype can be written as follows (deviations recorded in relation to a normal male complement): + l p - q + , +2, + 3 , + 4 q - , +5, + 5 , +7, +7, + 7 p - , +7p-, + Q - , +9p-, +lo, + l l q - , +13, +13, +14q+, +I%-, +Is, +16, +17, 18q-, +19, +19, +20, +21, +21, +Y, +Y, +X. Thus, the karyotype included 10 structurally changed chromosomes, i.e. markers. Only two of these had the same morphological features (mar ItI, cf. below). Each marker type is characterized in detail below: mar I
mar I1
+ : This big subtelocentric marker was clearly a chromosome No. 1 which had been subjected to a pericentric inversion; with regard to this mechanism and the location of the break points, the marker can be characterized by the formula inv (1) (p13q43). = 4q - : This small metacentric marker
mar
mar
mar
mar
= 1 p -q
mar
76 77
8
78
79
80 81100
*I50
8
5
3
4
1
100
was a chromosome No. 4 having lost the whole long arm except for a short proximal segment; the designation for the marker should be del (4) (q13). 111 = 7p - : The two markers of this type were chromosomes No. 7 having lost a small interstitial segment in the middle of the short arm; their designation should be del (7) ( ~ 1 3 ~ 1 5 ) . IV = 8q - : This marker was a chromosome No. 8 with shortened long arm due to an interstitial deletion; the designation for the anomaly should be del(8) (q21q23). V = 9p - :This marker was a chromosome No. 9 having lost the distal part of the short arm; the designation for the marker should be del (9) (p13). VI = l l q - : This marker was a chromosome No. 1 1 having lost a very small interstitial segment of the long arm; the designation for this anomaly should be del (1 1) (q22q23). VII = 14q +: This marker was a chromosome No. 14 having lost an interstitial segment in the middle of the long arm [del (14) (q22q23)I; a small, almost unstained segment of unknown origin had been inserted, t (14; ?) (q22-23; ?) VIII = 14q - : This marker was a chromosome No. 14 having lost the distal part of its long arm; the designation for the marker should be del (14) (q23). IX = 18q - : This marker was a chromosome No. 18 having lost approximately the distal third of its long arm; the designation for the marker should be del (18) (q22).
In slides stained with conventional methods, it Hrreditas 78, 1974
306
BRIEF REPORTS
2
1
3
i'
mar 1
5
4
6
7
8
Y+
4 4
mar
mar=
12
11
13
UI
14
15
Fig. I . The karyotype of a sternline cell i n 343 MG ( S = 75); mar= marker. x 2900.
Heretlitos 78, 1974
Pi
4
mar 31
BRIEF REPORTS
was possible to discern only one marker regularly, namely mar I. The other nine markers were hidden in groups C-G. The variant cells analyzed comprised seven cells with the S-number but having a deviating karyotype and 18 cells with perimodal chromosome numbers. All these cells were closely related to those with the S-karyotype. In the majority of the variant cells the differences were caused by gain or loss of one, rarely two, of one or several of the chromosomes Nos. 7, 1 1 , 12, 16 and 17 and/or gain or loss of marker chromosomes of the types I, 111, IV and VIII. Only a few cells contained new marker types; their origin was usually uncertain.
Discussion
The present established glioma cell line, 343 MG, had a hypertriploid mode, and the stemline karyotype included 10 structurally changed chromosomes, i.e. markers. The origin of all markers could be completely clarified except for the derivation of the inserted segment of mar VII (cf. above). At least eight of the 10 markers had resulted from interstitial or terminal deletions, and, as a rule, the deleted segments seemed to have been eliminated. These findings differ considerably from those in the two established glioma cell lines previously studied, namely 178 MG and 138 M G (MARKet al. 1974 a, b). Thus, in the hypodiploid (S = 45) 178 MG four of the eight markers were formed by reciprocal translocations of whole arms, one had resulted from a non-reciprocal translocation, and three from deletions. In the hypotriploid (S = 60-61) 138 M G four of the wholly indentified markers were isochromosomes, two others had resulted from non-reciprocal translocations, and one each from duplication and deletion of a segment. These observations indicate an individual structural evolution in each glioma cell line. There is, however, one similarity between the structural rearrangements in the different glioma cell lines, namely the preferential involvement of chromosomes in groups C and D. Accordingly, in 178 M G six out of eight markers were wholly or partly derived from C and/or D chromosomes, in 138 MG the same figures were seven out of the eight identified markers, and in 343 MG the corresponding figures were seven out of ten
307
markers. Within the two chromosome groups C and D, however, the pattern of involvement of different chromosome types was highly variable. The involvement of chromosome No. 7 is so far the only consistent festure in the three glioma cell lines studied. Already in the series of astrocytic gliomas studied in direct fixations and with conventional staining methods, there were indications that the chromosome groups C and D were prone to contribute to the formation of markers (MARK 1971). The findings in the glioma cell lines confirm this conclusion but the results also demonstrate the complex nature of the involvement of groups C and D. A complete evaluation of this preferential pattern can not be accomplished until more glioma cell lines have been analyzed. Those recently established and with their mode in the diploid region should be most suited for further studies. Acknowledgments. - The present work was supported by grants from the Swedish Cancer Society (Projects 209K74-06X, 55-872-07x8) and by a grant from the Foundation of the Carlsson family.
Department of Pathology, Central Hospital, Skovde and The Wallenberg Laboratory, Uppsala, Sweden
Literature cited MARK,J. 1971. Chromosomal characteristics of neurogenic tumours in adults. -- Hereditas 68: 61-100. -1974. Chromosomal patterns in benign and malignant human tumours in the nervous system. - In Chromosomes and Cancer (Ed. J. GERMAN),John Wiley, New York (In press I . MARK,J., P O N T ~ N J. ,and WESTERMARK, 8 . 1974a. G-band analyses of an established cell line of a human malignant glioma. - Humangenetik 22: 323-326. - 1974b. Origin of the marker chromosomes in an established hypotriploid glioma cell line studied with G band technique. - Acta Neuropathol. (In press). Paris Conference 1971. Standardization in Human Cytogenetics. -Birth Defects: Original Article Series, VIII: 7, 1972. The National Foundation, New York. PONT~N J ., and MACINTYRE, E. H. 1968. Long term cultureofnormal and neoplastic human glia. -Acta farhol. Microbiol. Scand. 74: 465-486. B., PONT~N, J. and HUGOSSON,R . 1973. WESTERMARK, Determinants for the establishment of permanent tissue Ibid. 81: 791-805. culture lines from human gliomas. -
Joachim Mark Department of Pathology Central Hospital of Skovde 541 01 Skovde, Sweden Hereditas 78, 1974
