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Regional mapping of the human immunoglobulin lambda light chain to the Philadelphia chromosome in chronic myeloid leukaemia
Leukemia Research Vol. 8, No. 4, pp. 547-553, 1984. Printed in Great Britain.
0145-2126/8453.00 +0.G0 © 1984 PerlUlmon Press Ltd.
REGIONAL MAPPING OF THE HUMAN IMMUNOGLOBULIN LAMBDA LIGHT CHAIN TO THE PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKAEMIA M.
H.
GOYNS*, B. D. YOUNG*, A. GEURTS VAN KESSEL~, A. DE KLEINt, G. GROSVELD~, C. R. BARTRAM~ AND D. BOOTSMA~" *Beatson Institute for Cancer Research, Garscube Estate, Glasgow, U.K. and tDept, of Clinical Genetics, Erasmus University, Rotterdam, Netherlands
(Received 25 October 1983. Accepted in final form 17 January 1984) Abstract--The lambda light chain immunoglobulin constant region (Ck) locus was.mapped on human chromosome 22. A DNA probe containing part of the Ck locus was isolated from a human chromosome 22 genomic library, and a series of rodent × human somatic cell hybrids (each of which contained different translocated parts of chromosome 22) were constructed and characterized. The hybridization of the Ck probe to DNA from these cell hybrids was then studied by Southern blot analysis. The results demonstrates that the C~. locus is situated very close to the translocation breakpoint on human chromosome 22 which is characteristic of chronic myeloid leukaemia, and at least part if not at all of the locus is situated on the Philadelphia chromosome.
Key words: lmmunoglobulin, chronic myeloid leukaemia, Philadelphia chromosome, translocation.
INTRODUCTION DURING the last two decades a number of acquired chromosomal abnormalities have been found to be specifically associated with particular human cancers [18, 23]. The occurrence of the Philadelphia (Ph t) translocation (9;22)(q34;ql 1) in patients with chronic myeloid leukaemia (CML) is one of the best documented examples of such an aberration [24]. The ql l-qter region of chromosome 22 is translocated to chromosome 9 [9], which results in 9q + , and 22q- derivative chromosomes, the latter being referred to as the Ph' chromosome. The reciprocity of this translocation has recently been confirmed by the demonstration [5] that the human cellular homologue (c-abl) of the transforming sequence of the Abelson murine leukaemic virus (A-MuLV) is translocated from chromosome 9 to chromosome 22q- (Table 1). Further, the cellular homologue of the simian sarcoma virus (c-sis) which is located on chromosome 22 [27], is included in the translocation to chromosome 9 [I1, 12] (Table I). Specific chromosomal rearrangements have also been reported in Burkitt's lymphoma and other B-cell neoplasms [16, 29]. The most common form is a translocation between chromosomes 8 and 14 (t(8;14)(q24;q32) with variant forms between 8 and 2 (t(2;8))(p12;q24) and between 8 and 22 (t(8;22))(q24;ql 1) occurring less frequently. Since chromosomes 14, 2 and 22 bear the heavy chain, kappa and lambda immunoglobulin gene clusters, respectively [4, 7, 21] it was proposed that genes critical for cellular transformation may be activated by juxtaposition after translocation to an active immunoglobulin locus [16, 25]. The localisation of the heavy chain [3, 15, 20]
Abbreviations: C2, immunoglobulin lambda light chain constant region; CML, chronic myeloid ieukaemia; Ph ', Philadelphia. Correspondence to: M. H. Goyns, Beatson Institute for Cancer Research, Garscube Estate, Glasgow, U.K. 547
548
M.H. GOYNSet al.
(14q32) and the kappa light chain [19] (2cen- 2p12) to the regions involved in chromosomal breakpoints reinforced this hypothesis. We report here the assignment of the lambda light chain (constant region) locus to the long arm of chromosome 22 at band ql l, the cytogenetic region in which breakpoints occur for both the 8;22 translocation of Burkitt's l y m p h o m a and the 9;22 translocation in CML. Furthermore our results show that at least part o f the constant region of the lambda locus remains on the Ph' chromosome in the 9;22 translocation. MATERIALS AND METHODS Cell culture
The somatic cell hybrids were constructed and characterized as previously described [8-10]. They were obtained by fusion of rodent cells with either human leucocytes carrying balanced reciprocal translocations (X;22)(q21;ql l) (hybrids 33-11 and 33-11TG) and (l;22)(q42ql3) (hybrids AM-27 and AM-21) or human CML cells carrying the Ph' transiocation (9;22)(q34;qll) (hybrids 14CB-SA, 14CB-21A, WESP-2A, PgMo-22 and ICB-17ANU). 1/22 AM and 33-11 hybrid clones were derived from fusion of human leucocytes carrying translocation 1;22 and X;22 with Chinese hamster a3 and E36 cells, repectively. Hybrids cell lines PgMe-25NU and PgMo-22were obtained after fusion of mouse Pgl9 cells with leucocytesfrom a normal human donor and a CML donor, respectively. WESP-2Aresults from fusion of mouse WEHI-3Bcells with leucocytesfrom a second CML patient. Leucocytesfrom a third CML patient were fused with Chinese hamster E36 cells to produce both 14CB clones and Chinese hamster cell line a3 was used to obtain ICB-17ANU(fourth CML patient). The cells were grown in Hams SF-12 medium supplemented with 10070foetal calf serum, in an atmosphere of 5;95070 Co2:air at 37°C. Hybridization analysis
High molecular weight DNA was prepared from the cell lines and cell hybrids [13]. Each of the DNA samples was restricted with EcoRl, size fractionated on a 1070agarosegel and transferred to nitrocellulose filters [26]The Chr22X clone, which contains the constant region structural genes Kern-OF and Kern-Oz+ was provided by Dr. T. H. Rabbitts. This was nicktranslated [22] to produce a probe with a specific activity of 10' cpm/v.g. After hybridization the filters were subjected to two 30-min washes in lxSSc and two 30-rain washes in 0.1SSC, 0.1 °70 SDS at 65°C. RESULTS A N D D I S C U S S I O N We have used a D N A probe from the lambda constant region to study D N A from cell hybrids bearing different translocated parts o f chromosome 22. The human C~, D N A probe, designated Chr22~,5 (Rabbitts and Matthews, in preparation) was obtained by screening a phage library o f chromosome 22 [17] with a mouse lambda constant region probe [l] and consisted of an 8kb Eco Rl fragment in ~,gt WES which included the two constant region structural genes, Kern - O z - a n d Kern -Oz + [14]. The identity of this clone was confirmed by restriction site mapping and by subcloning and sequencing of a part of one of its coding regions. D N A was prepared from the various cell lines, digested with Eco Rl and analysed by the Southern blotting technique using the nick translated Chr22~,5 probe. It can be seen from Fig.l that Chr22~,5 hybridizes mainly to normal human D N A (lane a) with an 8kb band which corresponds to the 8kb insert in our probe. Although this human D N A is from fibroblast material which is unrelated to any of the CML cell hybrids, it exhibits the same hybridization pattern that was observed with most of our human D N A samples (unpublished observation). This band is also observed in certain hybrid cells (Fig. l and Table l) An exception to this observation is the 15kb hybridizing band in the 22qcontaining cell line ICB -17ANU (lane m). This 15kb hybridizing band is due to an Eco Rl polymorph'ism as was checked by Hind III, Bgl lII and Bam H I digests of this cell line DNA. Eco Rl polymorphisms in the area of the genome covered by the Chr22~,5 probe have also been observed by others [14, 28]. It is also clear that there is no hybridization to the Chinese hamster (lanes b & e) or mouse parent cell DNAs under our stringency conditions. Hybrid clone PgMe-25NU has a normal chromosome as its only human component, while each of the other hybrids contains a few human chromosomes in addition to those relevent to the translocation. Positive hybridization of the Chr22~,5
a
b
c
d
e
f
g
h
k
I
FIB
n
FIG. 1. Segration of the constant region of the lambda immunoglobulin locus in rodent-human hybrids. (a) normal human fibroblast; (b), a3(hamster parentcell);(c), AM21; (d), AM27; (e), E36 (hamster parent cell); (f), 33-11; (g), 33-11TG; (h), WESP2A; (i), 14CB-$A; (j), 1aCB-21A; (k), PgMo-22; (1), PgMe-25NU; (m), ICB-17ANU; (n), WEHI-3B.
549
Mapping of lambda immunoglobulin locus TABLE
I.
SEGREGATION
c-abl a n d c-sis
OF HUMAN
ONCOGENES
AND
551 IMMUNOGLOBULIN
L A M B D A L I G H T C H A I N C O N S T A N T R E G I O N S E Q U E N C E S IN S O M A T I C C E L L H Y B R I D S C O N T A I N I N G
Ph'
Hybrid line
cell
Human 9
PgME-25NU IOCB-23B WESP-2A ICB-17ANU 14CB-5A 14CB-21A PgMo-22
chromosomes 22 9q + 22q-
+ + -
+ -
+ +
Oncogenes
c-abl*
c-sis'~
+ + + + -
+ + +
"+ + + -
lmmunoglobulin C h r 2 2 - ~.5
+ NT + + + -
* [ R e f . 51. ~'[Refs. 11, 12]. NT, Not tested.
probe to the DNA of PgMe-25NU (Fig. 1, lane 1) confirms the localization of the lambda light chain locus to chromosome 22. Figure 1 shows that C~. genes could also be detected in DNA from cells AM-27 (lane d), 33-11 (lane f), 33-11TG (lane g), WESP-2A (lane h), 14CB-5A (lane i) and I CB-17ANU (lane m). None of these cell lines has a normal human chromosome 22. Figure 2 shows diagrammatically the regions of chromosome 22 which have been retained by the different hybrid cell lines and the respective Chr22k5 hybridization data. It is evident from the data that the constant part of the lambda locus lies within band 22q I 1 in a region bounded by the chromosomal breakpoints producing the X;22 and 9;22 translocations (designated the shortest region of overlap, SRO, in Fig. 2). The breakpoint on chromosome 22 has been reported [8] to be just below the
X;22
22 PgMo -~I6NU
4-
22
1;22
,b
13 p
9;22(CML)
33-114-133"11TG4-AM~:llAM'2--1"t"
12 11
11 q
~RO" "
. . . . . .
q ~ d~
12 13
r _ _
_
I
FIG. 2. S c h e m a t i c r e p r e s e n t a t i o n o f t h e d i s t r i b u t i o n o f t r a n s l o c a t e d p a r t s o f c h r o m o s o m e 2 2 i n t h e s o m a t i c cell h y b r i d s . C h r o m o s o m e analysis was done using reverse banding with acridine orange, a f t e r h e a t d e n a t u r a t i o n . A t l e a s t 16 m e t a p h a s e s w e r e a n a l y s e d p e r cell l i n e . E n z y m e m a r k e r o f t h e h y b r i d s h a s b e e n r e p o r t e d p r e v i o u s l y ( G u e r t s v a n K e s s e l et al., 1980, 1981 ; d e K l e i n et al., 1983) + indicates hybridization of the Chr22k5 probe. SRO indicates the shortest region of overlap: the r e g i o n o f c h r o m o s o m e 22 t h a t c o n t a i n s t h e l a m b d a l i g h t c h a i n c o n s t a n t r e g i o n s e q u e n c e s .
552
M . H . GOYNS et al.
centromere in the X;22 translocation and, therefore, appears to be proximal to that observed in the 9;22 translocation. This is in agreement with our present analysis. The question of whether the breakpoint occurs within the C~. locus remains unanswered, even though DNA from cells containing the 9q ~, without 22q-, fail to show hybridization to the Chr22Z5 probe (Fig. 1, lanes j and k): this latter evidence may be misleading since the DNA from these hybrid cells hybridizes human probes weakly and because the Chr223.5 probe is not completely homologous to the other C3. genes which might be translocated. Furthermore, the experiment shown in Fig. l has not resolved the 14kb and 16kb EcoRl fragments which come from either side of the 8kb region contained in Chr22Z5 [14]. We cannot, therefore, be certain whether one or both of these fragments exist in the Ph ' positive cell hybrid DNA. Since hybrid cell lines derived from four different leukaemia patients were used, we conclude that the observed localization of C~. sequences is most probably a general phenomenon in the Ph i translocation in CML. The present state of the experiments using C~, c-abl, and c-sis probes with normal or CML derived DNA is summarised in Table I. It is clear that as a result of the 9;22 translocation the c-abl gene and the lambda constant region become linked on the Philadelphia chromosome. We do not yet know the distance between C~, and c-abl on the Ph i chromosome and this analysis must await cloning of the relevent segments from CML DNA. However, recent evidence has indicated that both C3~ and c-abl genes are amplified in the CML cell line, K562 [2] which suggests that they may be closely linked. Recently, it has been found [6] that human lambda constant region sequences are translocated to chromosome 8 in the Burkitt lymphoma associated translocation (8;22)(q24;q I 1) using in situ hybridization techniques. Clear evidence for translocation of C~. sequences was not observed in the CML patients studied here, indicating that the breakpoint in chromosome 22 in the Ph i translocation may be distal to that observed in the Burkitt lymphoma associated translocation t(8;22). Acknowledgements--The authors wish to thank Dr. A. Hagemeijer and Dr. W. Witterland for their support and Mr. A. J. van Agthoven for expert technical assistance. This work has been supported by grants from the Leukaemia Research Fund, the Cancer Research Campaign, the Medical Research Council and the Netherlands Cancer Society (Koningin Wilhelmina Fonds). C.R.B. is a recipient of a fellowship from the Deutsche Forschungsgemeinscha ft.
REFERENCES l. BOTHWELLA. L. M., PASKIND M., SCHWARTZ R. C., SONENSHEIN G. E., GEFTER M. L. & BALTIMORE D. (1981) Dual expression of k genes in the MOPC-315 plasmacytoma. Nature 290, 65. 2. COLLINS S. J & GROUDINE M. T. 0983) Rearrangement and amplification of c-agl sequences in the human chronic myeiogenous luekaemic cell line K562. Proc. hath. Aead. Sci. U.S.A. 80, 4813. 3. Cox D. W., MARKOVIC V. D. & TESHIMA I.E. (1982) Genes for immunoglobulin heavy chains and for ce,antitrypsin are localized to specific regions of chromosome 14q. Nature 297, 430. 4. CROCE C. M., SHANDER M., MARTINIS J., CICUREL L., D'ANCONA (3. G., DOLBY T. W. & KEPROWSKI H. (1979) Chromosomal localization of the genes for human immunoglobulin heavy chains. Proc. hath. Acad. Sci. U.S.A. 76,3416. 5. DEKLEIN A., GEURTSVAN KESSEL A., GROSVELDG., BARTRAMC. R., HAGEMEIJERA., BOOTSMAD., SPURR N. K., HEISTERKAMPN., GROFFEN J. '£" STEPHENSON J.R. (1982) A cellular oncogene (c-abl) is translocated to the Philadelphia chromosome in chronic myeloid leukaemia. Nature 300, 765. 6. DE LA CHAPELLE A., LENOIR G., BOUE J., BOUE A., GALLANO P., HUERRE C., SZAJNERT M. F., JEANPlERRE M., LALOUEL J. M.,&. KAPLAN J. C. (1983) Lambda lg constant region genes are translocated to chromosome 8 in Burkitts lyrnphoma with t(8:22) Nucl Acid Res. 11, 1133. 7. ERIKSON J., MARTINIS J. & CROCE C. M. (1981) Assignment of the genes for human ~ immunoglobulin chains to chromosome 22. Nature 294, 173. 8. GEURTSVAN KESSEL A., TETTEROO P. A. T., VON DEM BORNE A. E. G. K., HAGEMEIJERA. & BOOTSMAD. (1983) Expression of human myeloid association surface antigens in human-mouse myeloid cell hybrids Proc. hath. Acad. Sci. U.S.A. gO, 3748. 9. GEURTSVAN KESSEL A., TIENBRINKE H., BOERE W. A. M., DER BOER W. C., DEGROOT P. G., HAGEMEIJER A., MEERA KHAN P. & PEARSON P. L. (1981) Characterization of the Philadelphia chromosome by gene mapping. Cytogenet Cell Genet 30, 83.
Mapping of lambda immunoglobulin locus
553
10. GEURTS VAN KESSEL A., WESTERVELD A., DEGROOT P. G., MEERA KHAN P. & HAGEMEIJER A. (1980) Regional localization of the genes coding for human ACO2, ARSA and NAGA on chromosome 22. Cytogenet. Cell Genet. 28, 169. 11. GOYNSM. H., GEURTSVAN KESSELA., GALLO R. C., RABBITTST., MACKENZIEE. D. il~ YOUNGB. D. (1983) Mapping of the immunoglobulin lambda light chain constant region locusand the c-sis onogene on chromosome 22. Fur. J Cell Biol 30 supp 1, 19. 12. GROFFEN J., HEIgTERKAMP N., STEPHENSON J. R., (3EURTS VAN KESSEL A., DEKLEIN A, GROSVELD (3. BOOTSMA D. (1983) c-sis is transiocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia. J. exp. Med. 158, 9. 13. GROss-BELLARD M., OUDET P. & CHAMBON P (1973) Isolation of high-molecular-weight DNA from mammalian cells. Eur. J Biochem 36, 32. 14. HIETER P. A., HOLUS (3. F., KORSMEYER S. J., WALDMANN T. H. & LEVER P. (1981) Clustered arrangements of immunoglobulin X constant regions. Nature 294, 536. 15. KIRSCr~1. R., MORTON C. C., NAKAHARA,K. & LEVER P. (1982) Human immunoglobulin heavy chain genes map to a region of translocations in malignant B iymphocytes. Science 216, 301. 16. KLEIN (3. (1981) The role of gene dosage and genetic transpositions in carcinogenesis. Nature 294, 313. 17. KRUMLAUF R., JEANPIERRE M. & YOUNG B. D. (1982) Construction and characterization of genomic libraries from specific human chromosomes. Proc. natn. Acad. Sci. U.S.A. 79 2971. 18. LAWLER S. D. (1977) The cytogenetics of chronic granuiocytic leukaemia Ciin. Haemat. 6, 55. 19. MALCOLM S., BARTON P., MURPHY C., FERGUSON-SMITH M. A., BENTLY D. L. & RABm-r'rs T. H. (1982) Localization of human immunoglobulin K light chain variable region genes to the short arm of chromosome 2 by in situ hybridization Proc. natn. Acad. Sci. U.S.A. 79, ,1957. 20. MCBRIDE O. W., BATTEY J., HOLLIS (3. F., SWAN D. C., SIEBENUSTU& LEDER P., (1982) Localization of the human variable and constant region immunoglobulin heavy chain genes subtelomeric band q32 of chromosome 14. Nucl Acid Res. 10, 8155. 21. MCBRIDE O. W., HIE'rER P. A., HOLLIS (3. F., SWAN D., OTEY M. C. & LEDER P., (1982) Chromosomal localization of human kappa and lambda immunoglobulin light chain constant region genes. J. exp. Med. 155, M80. 22. RIGBY P. J. W., DIECKMANN M., RHODES C. & BERG P. (1977) Labelling deoxyribonucleic acid to a high specific activity in vitro by nick translation with DNA polymerase. J molec. Biol 113, 237. 24. ROWLEY J. D., (1980) Ph'-positive leukaemia, including chronic myelogenous ieukaemia. Clin Haemat 9, 55. 24. ROWLEY J. D., (1980) Ph'-positive leukaemia, including chronic myeloigenous leukaemia. Clin Haemat 9, 55. 25. ROWLEY J. D., (1982) Identification of the constant chromosome regions involved in human haematologic malignant disease. Science 216, 749. 26. SOUTHERN E. D., (1973) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 9g, 503. 27. SWAN D. C., MCBRIDE O. W., ROBmNS K. C., KE1TMLEY D. A., REDDY E. P. & AARONSON S. A. (1982) Chromosmal mapping of the simian sarcoma virus one gene analogue in human cells. Proc. natn. Acad. Sci. U.S.A. 79, 4691. 28. TAuB R. A., HOLUS G. F., HIETER P. A., KORSMEYER S., WAt.DMANN T. A. & LEDER P. (1983) Variable amplification of immunoglohulin X light-chain genes in human populations. Nature 304, 172. 29. ZECH L., MAGLUND U., NILSSON K. & KLEIN (3. (1976) Characteristic chromosomal abnormalities in biopsies and lymphoid cell lines from patients with Burkitt and non-Burkitt lymphoma. Int. J. Cancer 17, 47.
0145-2126/8453.00 +0.G0 © 1984 PerlUlmon Press Ltd.
REGIONAL MAPPING OF THE HUMAN IMMUNOGLOBULIN LAMBDA LIGHT CHAIN TO THE PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKAEMIA M.
H.
GOYNS*, B. D. YOUNG*, A. GEURTS VAN KESSEL~, A. DE KLEINt, G. GROSVELD~, C. R. BARTRAM~ AND D. BOOTSMA~" *Beatson Institute for Cancer Research, Garscube Estate, Glasgow, U.K. and tDept, of Clinical Genetics, Erasmus University, Rotterdam, Netherlands
(Received 25 October 1983. Accepted in final form 17 January 1984) Abstract--The lambda light chain immunoglobulin constant region (Ck) locus was.mapped on human chromosome 22. A DNA probe containing part of the Ck locus was isolated from a human chromosome 22 genomic library, and a series of rodent × human somatic cell hybrids (each of which contained different translocated parts of chromosome 22) were constructed and characterized. The hybridization of the Ck probe to DNA from these cell hybrids was then studied by Southern blot analysis. The results demonstrates that the C~. locus is situated very close to the translocation breakpoint on human chromosome 22 which is characteristic of chronic myeloid leukaemia, and at least part if not at all of the locus is situated on the Philadelphia chromosome.
Key words: lmmunoglobulin, chronic myeloid leukaemia, Philadelphia chromosome, translocation.
INTRODUCTION DURING the last two decades a number of acquired chromosomal abnormalities have been found to be specifically associated with particular human cancers [18, 23]. The occurrence of the Philadelphia (Ph t) translocation (9;22)(q34;ql 1) in patients with chronic myeloid leukaemia (CML) is one of the best documented examples of such an aberration [24]. The ql l-qter region of chromosome 22 is translocated to chromosome 9 [9], which results in 9q + , and 22q- derivative chromosomes, the latter being referred to as the Ph' chromosome. The reciprocity of this translocation has recently been confirmed by the demonstration [5] that the human cellular homologue (c-abl) of the transforming sequence of the Abelson murine leukaemic virus (A-MuLV) is translocated from chromosome 9 to chromosome 22q- (Table 1). Further, the cellular homologue of the simian sarcoma virus (c-sis) which is located on chromosome 22 [27], is included in the translocation to chromosome 9 [I1, 12] (Table I). Specific chromosomal rearrangements have also been reported in Burkitt's lymphoma and other B-cell neoplasms [16, 29]. The most common form is a translocation between chromosomes 8 and 14 (t(8;14)(q24;q32) with variant forms between 8 and 2 (t(2;8))(p12;q24) and between 8 and 22 (t(8;22))(q24;ql 1) occurring less frequently. Since chromosomes 14, 2 and 22 bear the heavy chain, kappa and lambda immunoglobulin gene clusters, respectively [4, 7, 21] it was proposed that genes critical for cellular transformation may be activated by juxtaposition after translocation to an active immunoglobulin locus [16, 25]. The localisation of the heavy chain [3, 15, 20]
Abbreviations: C2, immunoglobulin lambda light chain constant region; CML, chronic myeloid ieukaemia; Ph ', Philadelphia. Correspondence to: M. H. Goyns, Beatson Institute for Cancer Research, Garscube Estate, Glasgow, U.K. 547
548
M.H. GOYNSet al.
(14q32) and the kappa light chain [19] (2cen- 2p12) to the regions involved in chromosomal breakpoints reinforced this hypothesis. We report here the assignment of the lambda light chain (constant region) locus to the long arm of chromosome 22 at band ql l, the cytogenetic region in which breakpoints occur for both the 8;22 translocation of Burkitt's l y m p h o m a and the 9;22 translocation in CML. Furthermore our results show that at least part o f the constant region of the lambda locus remains on the Ph' chromosome in the 9;22 translocation. MATERIALS AND METHODS Cell culture
The somatic cell hybrids were constructed and characterized as previously described [8-10]. They were obtained by fusion of rodent cells with either human leucocytes carrying balanced reciprocal translocations (X;22)(q21;ql l) (hybrids 33-11 and 33-11TG) and (l;22)(q42ql3) (hybrids AM-27 and AM-21) or human CML cells carrying the Ph' transiocation (9;22)(q34;qll) (hybrids 14CB-SA, 14CB-21A, WESP-2A, PgMo-22 and ICB-17ANU). 1/22 AM and 33-11 hybrid clones were derived from fusion of human leucocytes carrying translocation 1;22 and X;22 with Chinese hamster a3 and E36 cells, repectively. Hybrids cell lines PgMe-25NU and PgMo-22were obtained after fusion of mouse Pgl9 cells with leucocytesfrom a normal human donor and a CML donor, respectively. WESP-2Aresults from fusion of mouse WEHI-3Bcells with leucocytesfrom a second CML patient. Leucocytesfrom a third CML patient were fused with Chinese hamster E36 cells to produce both 14CB clones and Chinese hamster cell line a3 was used to obtain ICB-17ANU(fourth CML patient). The cells were grown in Hams SF-12 medium supplemented with 10070foetal calf serum, in an atmosphere of 5;95070 Co2:air at 37°C. Hybridization analysis
High molecular weight DNA was prepared from the cell lines and cell hybrids [13]. Each of the DNA samples was restricted with EcoRl, size fractionated on a 1070agarosegel and transferred to nitrocellulose filters [26]The Chr22X clone, which contains the constant region structural genes Kern-OF and Kern-Oz+ was provided by Dr. T. H. Rabbitts. This was nicktranslated [22] to produce a probe with a specific activity of 10' cpm/v.g. After hybridization the filters were subjected to two 30-min washes in lxSSc and two 30-rain washes in 0.1SSC, 0.1 °70 SDS at 65°C. RESULTS A N D D I S C U S S I O N We have used a D N A probe from the lambda constant region to study D N A from cell hybrids bearing different translocated parts o f chromosome 22. The human C~, D N A probe, designated Chr22~,5 (Rabbitts and Matthews, in preparation) was obtained by screening a phage library o f chromosome 22 [17] with a mouse lambda constant region probe [l] and consisted of an 8kb Eco Rl fragment in ~,gt WES which included the two constant region structural genes, Kern - O z - a n d Kern -Oz + [14]. The identity of this clone was confirmed by restriction site mapping and by subcloning and sequencing of a part of one of its coding regions. D N A was prepared from the various cell lines, digested with Eco Rl and analysed by the Southern blotting technique using the nick translated Chr22~,5 probe. It can be seen from Fig.l that Chr22~,5 hybridizes mainly to normal human D N A (lane a) with an 8kb band which corresponds to the 8kb insert in our probe. Although this human D N A is from fibroblast material which is unrelated to any of the CML cell hybrids, it exhibits the same hybridization pattern that was observed with most of our human D N A samples (unpublished observation). This band is also observed in certain hybrid cells (Fig. l and Table l) An exception to this observation is the 15kb hybridizing band in the 22qcontaining cell line ICB -17ANU (lane m). This 15kb hybridizing band is due to an Eco Rl polymorph'ism as was checked by Hind III, Bgl lII and Bam H I digests of this cell line DNA. Eco Rl polymorphisms in the area of the genome covered by the Chr22~,5 probe have also been observed by others [14, 28]. It is also clear that there is no hybridization to the Chinese hamster (lanes b & e) or mouse parent cell DNAs under our stringency conditions. Hybrid clone PgMe-25NU has a normal chromosome as its only human component, while each of the other hybrids contains a few human chromosomes in addition to those relevent to the translocation. Positive hybridization of the Chr22~,5
a
b
c
d
e
f
g
h
k
I
FIB
n
FIG. 1. Segration of the constant region of the lambda immunoglobulin locus in rodent-human hybrids. (a) normal human fibroblast; (b), a3(hamster parentcell);(c), AM21; (d), AM27; (e), E36 (hamster parent cell); (f), 33-11; (g), 33-11TG; (h), WESP2A; (i), 14CB-$A; (j), 1aCB-21A; (k), PgMo-22; (1), PgMe-25NU; (m), ICB-17ANU; (n), WEHI-3B.
549
Mapping of lambda immunoglobulin locus TABLE
I.
SEGREGATION
c-abl a n d c-sis
OF HUMAN
ONCOGENES
AND
551 IMMUNOGLOBULIN
L A M B D A L I G H T C H A I N C O N S T A N T R E G I O N S E Q U E N C E S IN S O M A T I C C E L L H Y B R I D S C O N T A I N I N G
Ph'
Hybrid line
cell
Human 9
PgME-25NU IOCB-23B WESP-2A ICB-17ANU 14CB-5A 14CB-21A PgMo-22
chromosomes 22 9q + 22q-
+ + -
+ -
+ +
Oncogenes
c-abl*
c-sis'~
+ + + + -
+ + +
"+ + + -
lmmunoglobulin C h r 2 2 - ~.5
+ NT + + + -
* [ R e f . 51. ~'[Refs. 11, 12]. NT, Not tested.
probe to the DNA of PgMe-25NU (Fig. 1, lane 1) confirms the localization of the lambda light chain locus to chromosome 22. Figure 1 shows that C~. genes could also be detected in DNA from cells AM-27 (lane d), 33-11 (lane f), 33-11TG (lane g), WESP-2A (lane h), 14CB-5A (lane i) and I CB-17ANU (lane m). None of these cell lines has a normal human chromosome 22. Figure 2 shows diagrammatically the regions of chromosome 22 which have been retained by the different hybrid cell lines and the respective Chr22k5 hybridization data. It is evident from the data that the constant part of the lambda locus lies within band 22q I 1 in a region bounded by the chromosomal breakpoints producing the X;22 and 9;22 translocations (designated the shortest region of overlap, SRO, in Fig. 2). The breakpoint on chromosome 22 has been reported [8] to be just below the
X;22
22 PgMo -~I6NU
4-
22
1;22
,b
13 p
9;22(CML)
33-114-133"11TG4-AM~:llAM'2--1"t"
12 11
11 q
~RO" "
. . . . . .
q ~ d~
12 13
r _ _
_
I
FIG. 2. S c h e m a t i c r e p r e s e n t a t i o n o f t h e d i s t r i b u t i o n o f t r a n s l o c a t e d p a r t s o f c h r o m o s o m e 2 2 i n t h e s o m a t i c cell h y b r i d s . C h r o m o s o m e analysis was done using reverse banding with acridine orange, a f t e r h e a t d e n a t u r a t i o n . A t l e a s t 16 m e t a p h a s e s w e r e a n a l y s e d p e r cell l i n e . E n z y m e m a r k e r o f t h e h y b r i d s h a s b e e n r e p o r t e d p r e v i o u s l y ( G u e r t s v a n K e s s e l et al., 1980, 1981 ; d e K l e i n et al., 1983) + indicates hybridization of the Chr22k5 probe. SRO indicates the shortest region of overlap: the r e g i o n o f c h r o m o s o m e 22 t h a t c o n t a i n s t h e l a m b d a l i g h t c h a i n c o n s t a n t r e g i o n s e q u e n c e s .
552
M . H . GOYNS et al.
centromere in the X;22 translocation and, therefore, appears to be proximal to that observed in the 9;22 translocation. This is in agreement with our present analysis. The question of whether the breakpoint occurs within the C~. locus remains unanswered, even though DNA from cells containing the 9q ~, without 22q-, fail to show hybridization to the Chr22Z5 probe (Fig. 1, lanes j and k): this latter evidence may be misleading since the DNA from these hybrid cells hybridizes human probes weakly and because the Chr223.5 probe is not completely homologous to the other C3. genes which might be translocated. Furthermore, the experiment shown in Fig. l has not resolved the 14kb and 16kb EcoRl fragments which come from either side of the 8kb region contained in Chr22Z5 [14]. We cannot, therefore, be certain whether one or both of these fragments exist in the Ph ' positive cell hybrid DNA. Since hybrid cell lines derived from four different leukaemia patients were used, we conclude that the observed localization of C~. sequences is most probably a general phenomenon in the Ph i translocation in CML. The present state of the experiments using C~, c-abl, and c-sis probes with normal or CML derived DNA is summarised in Table I. It is clear that as a result of the 9;22 translocation the c-abl gene and the lambda constant region become linked on the Philadelphia chromosome. We do not yet know the distance between C~, and c-abl on the Ph i chromosome and this analysis must await cloning of the relevent segments from CML DNA. However, recent evidence has indicated that both C3~ and c-abl genes are amplified in the CML cell line, K562 [2] which suggests that they may be closely linked. Recently, it has been found [6] that human lambda constant region sequences are translocated to chromosome 8 in the Burkitt lymphoma associated translocation (8;22)(q24;q I 1) using in situ hybridization techniques. Clear evidence for translocation of C~. sequences was not observed in the CML patients studied here, indicating that the breakpoint in chromosome 22 in the Ph i translocation may be distal to that observed in the Burkitt lymphoma associated translocation t(8;22). Acknowledgements--The authors wish to thank Dr. A. Hagemeijer and Dr. W. Witterland for their support and Mr. A. J. van Agthoven for expert technical assistance. This work has been supported by grants from the Leukaemia Research Fund, the Cancer Research Campaign, the Medical Research Council and the Netherlands Cancer Society (Koningin Wilhelmina Fonds). C.R.B. is a recipient of a fellowship from the Deutsche Forschungsgemeinscha ft.
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