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Chromosome analysis of human neuroblastoma cell line TR14 showing double minutes and an aberration involving chromosome 1
Chromosome Analysis of Human Neuroblastoma Cell Line TR14 Showing Double Minutes and an Aberration Involving Chromosome 1 John K. Cowell and H. Thomas Rupniak
ABSTRACT:
The chromosomal analysis of a human neuroblastoma cell line derived from a pediatric patient is presented. The cell line has a modal chromosome number of 64 and contains large numbers of double minutes (DMs). The DMs are stable over many passages in vitro. A consistent feature of the karyotype is an abnormality involving the short arm of chromosome 1, which is consistent with previous reports from human neuroblastomas. The region distal to band lp31 has homogeneously staining characteristics and as such this cell line represents a rare example where DMs and HSRs are constitutive features of the same cells. The possibility that such regions could arise from chromosome rearrangements is discussed.
INTRODUCTION The belief that specific c h r o m o s o m e aberrations m a y be involved in the p r e d i s p o sition to the d e v e l o p m e n t of cancer has been strongly s u p p o r t e d by the findings in several neoplasms of pediatric origin. In two cases the increased probability of tumor d e v e l o p m e n t has been correlated with the inheritance or spontaneous dev e l o p m e n t of small, specific c h r o m o s o m e deletions. The risk of d e v e l o p i n g retinoblastoma has been associated with the deletion of b a n d 13q14 [1] and the deletion of c h r o m o s o m e b a n d 11p13 greatly e n h a n c e d the chance of d e v e l o p i n g Wilms' tumor [2]. Neuroblastomas represent another relatively c o m m o n pediatric cancer w h i c h shows an inherited p r e d i s p o s i t i o n [3]. A c c u m u l a t e d evidence has revealed the association of three specific c h r o m o s o m e abnormalities: aberrations involving the short arm of c h r o m o s o m e 1, double minutes (DMs), and h o m o g e n e o u s l y staining regions (HSRs). Although DMs and HSRs have been reported in tumors from a variety of different cell types, neuroblastomas probably represent the most frequent example [4]. In this report we present the cytogenetic findings from h u m a n neuroblastoma cell line TR14, derived from a pediatric patient, w h i c h contains DMs and an aberration involving the short arm of c h r o m o s o m e 1 w h i c h is possibly an HSR.
From the Department of Haematologyand Oncology,Institute of Child Health, London (J.K.C.)and the Department of Cellular Chemotherapy, Imperial Cancer Research Fund, London. Address requests for reprints to John K. Cowell, Department of Haematology and Oncology, Institute of Child Health, 30 Guilford Street, London WC1, England. Received October 13, 1982; accepted December 1, 1982.
273 © Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 0165-4608/83/$03.00
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J.K. Cowell et al.
MATERIAL AND METHODS
Cells and Cell Culture The TR14 cell line was derived from biopsy material of a 3-year-old male with stage III disease who had previously been treated with chemotherapy--Vincristine, Cyclophosphamide, Adriamycin, Melphalan (high-dose)--and low-dose radiotherapy. The procedure for isolation of the cell line and the characteristics of the resultant cells have been presented in more detail elsewhere [5]. All ceils were grown in Hams F12 medium supplemented with 10% fetal calf serum and transferred and harvested with trypsin. Chromosome Preparations The cell cycle time of the TR14 cells was approximately 3 days which made it difficult to obtain large numbers of metaphases without excessive treatment with mitotic arresting agents which would cause chromosome contraction. A 2-hr vinblastine treatment proved to be the maximum acceptable, after which the cells were
Figure 1
Representative Giemsa-banded karyotype from the TR14 cell line showing three
copies of the abnormality on chromosome 1.
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harvested and chromosome preparations made using standard air drying techniques [6]. T r y p s i n - G i e s m a b a n d i n g was carried out according to a modified technique described previously [6]. RESULTS In this study 20 well-banded metaphases were analyzed, giving the modal chromosome n u m b e r in TR14 cells as 63 (54%), with a range of 60-64. Sporadic chromosome counts over a 12-month period showed this n u m b e r to be fairly constant during in vitro culture. A representative karyotype of the TR14 cell line is s h o w n in Figure 1. Chromosomal variation among cells could be accounted for by apparently r a n d o m losses and gains of i n d i v i d u a l chromosomes. A n HSR-like region was present on the short arm of chromosome 1 distal to band lp31 (Fig. 2) and the tip had acquired a heavy band. In the majority of cells there were at least two of these markers and sometimes three. Four other chromosome markers were present (Fig. 1), one of w h i c h - - m l - - i n v o l v e s the translocation of the long arm of chromosome 3 onto the long arm of chromosome 1 (Fig. 1) and as such was similar to a marker seen in HeLa cells [7]. The other three markers could not be positively identified. It was possible that the distal region of the chromosome 1 marker was not an HSR but had arisen as a result of chromosome rearrangement (see Discussion). The distal region of l p is normally weakly banded (Fig. 2) and a rearrangement may
Figure 2 (Top) The homogeneously staining region on the short arm of chromosome 1 in TR14. (Bottom) A normal chromosome 1 (left) is compared with the abnormal 1 (right) and illustrates how the homogeneously staining region (HSR) might have arisen from the translocation of an unidentified chromosome region to the tip of the short arm of chromosome 1. A suggested breakpoint is indicated by the arrow.
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J.K. Cowell et al. involve the relocation of an unidentified region to the tip of l p with a breakpoint in lp36. The presence of the distal heavy band suggests that some translocation had occurred although the identity of the material could not be determined.
Double Minutes In the TR14 cell line there were often i n n u m e r a b l e double minutes (Fig. 3, 4) in every cell. T y p i c a l l y they varied in n u m b e r from cell to cell, sometimes c o m p l e t e l y covering the c h r o m o s o m e s (Fig. 3). Karyotypes m a d e 12 months apart i n d i c a t e d that the DMs have persisted in the cultured cells for at least this period of time. Not only was there heterogeneity in DM n u m b e r but size heterogeneity between DMs was also noted in the same cell (Fig. 5). The size of DMs in the TR14 cell line varied from the limits of resolution to relatively large DMs (Fig. 5). The presence of DMs and the HSR-like region in l p was noted in all cells. DISCUSSION The short arm of c h r o m o s o m e 1 is frequently involved in c h r o m o s o m e abnormalities in both neuroblastoma tissue and in cell lines derived from t h e m [8]. Consistent with this general finding, neuroblastoma cell line TR14 also has an aberration involving the short arm of chromosome 1. Deletion of most or part of the short arm has been the most consistent finding [8-11], the deleted region in c o m m o n to t h e m all being that distal to band lp32. In one of these cases this deletion represented the only c h r o m o s o m e abnormality in the cell [9]. In some cases where there was a p p a r e n t l y no deletion of chromosomal material, the short arm of c h r o m o s o m e 1 could be either relocated in the genome [10], or be the recipient site for a translocation or insertion [8]. These findings further i m p l y that disturbance of the genetic material in the short arm of chromosome 1 m a y be important in the d e v e l o p m e n t of neuroblastoma. Many neuroblastomas are n e a r - d i p l o i d [8]; TR14 however was subtetraploid, p r e s u m a b l y as a result of tetraploid formation and c h r o m o s o m e loss, a feature of some other established neuroblastoma cell lines [12]. The presence of two or more copies of the abnormal c h r o m o s o m e 1 suggests that this a b n o r m a l i t y was present in the cell line before endoreduplication, in contrast to the other c h r o m o s o m e markers in TR14 w h i c h were only present in single copy. Other c h r o m o s o m e abnormalities found frequently in neuroblastomas are HSRs [10] where they have been shown to be associated with m a n y different chromosomes. W h e n the HSR was located on c h r o m o s o m e 1 its position was variable, although in one case (IMR 32) it was located w i t h i n the frequently deleted 1p32ter region [11]. In TR14 the aberrant c h r o m o s o m e 1 was cytologically similar to that reported in IMR 32, an i n d e p e n d e n t l y derived cell line [14]. The same HSR w i t h a subterminal pale region and a distally located heavy b a n d was also observed in a retinoblastoma cell line [15]. There is now strong evidence that, in a few specific systems, HSRs represent gene amplification [16-18] and, by analogy, it is generally considered that all other HSRs also represent gene amplification [4]. Cytologically the subterminal region of chromosome 1 in TR14 is characteristic of an HSR but w h e t h e r it has arisen as a result of localized gene amplification or chromosome rearrangement is uncertain. One difficulty in distinguishing between these two possibilities lies in the fact that the l p 3 2 - t e r region is ordinarily faintly banded. Relocation of similar regions "back-to-back" could give the i m p r e s s i o n of an HSR. The presence of a distally located heavy band, not n o r m a l l y present on chromosome 1, further suggests that a rearrangement has occurred at some stage. Such a rearrangement possibly involves
Figure 3 Figure 4
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Giemsa-stained metaphase spread from TR14 showing large numbers of DMs with variable size. A cell from TR14 with fewer but more homogeneously sized DMs (arrows).
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Figure 5 Heterogeneity in DM size within a single cell from TR14. DMs vary from the limits of resolution to a size (arrows) approaching that of the smallest human chromosome.
one of the breakpoints in band l p 3 6 and is thus similar to that in a previous report by Brodeur et al. [9]. It was not possible in this case to determine whether there had been any deletion of chromosomal material during the evolution of the abnormal chromosome. The only detectable short arm material from chromosome 1 was present on the marker chromosome, which possibly makes any large deletion unlikely as this w o u l d represent complete loss of this material from the cell. The constitutive presence of DMs and HSRs in the same cell is rare [4] which is another reason to conclude that the u n b a n d e d region i n TR14 is not an HSR. A n alternative possibility is that two completely different genes have been amplified and in these cells the amplification has been mediated by DM and HSR formation, respectively. The TR14 cell line was not resistant to methotrexate and the identity of the amplified genes is not known. It is also completely u n k n o w n whether the amplified sequences are related to the malignancy of the cells. It may be that there are several different genes present in the DMs. The answers to these questions might be obtained from specific DM-DNA clones from TR14, which w o u l d be a suitable starting material because of the high n u m b e r of DMs per cell and their stability in the cells in vitro. Since neuroblastomas often contain DMs [10] an analysis of this type might prove useful in determining whether the same genes are being amplified in other h u m a n neuroblastomas.
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The frequent presence of DMs and HSRs in h u m a n neuroblastomas suggests that this tumor cell type shows a propensity for the genesis of amplified sequences [19]. Whether these genes are involved in m a l i g n a n c y of the cell is not known. It might be that they represent a genetic imbalance i n the cell responsible for the malignant phenotype. It was suggested by Sachs and co°workers [20, 21] that cancer might be due to chromosome imbalance, defined as a change in the relative dose of certain genes in malignant cells compared with the normal cell. Chromosome imbalance can possibly be generated in m a n y ways [22, 23] i n c l u d i n g deletions and duplications of whole or parts of chromosomes, tetraploid formation and chromosome loss [24, 25], and also from position effect due to chromosome rearrangement [22]. All of these chromosome abnormalities at one time or another have been described associated with chromosome 1 in neuroblastomas [8]. It is possible therefore that imbalance for the 1p32-ter region is important in the genesis of neuroblastoma. Theoretically imbalance can also be produced by specific amplification of the gene(s) relevant to m a l i g n a n c y by the formation of HSRs and DMs. In one particular cell line an HSR has been shown to be located w i t h i n the 1p32-ter region [13]. Since DMs are an alternative form of HSRs [4], it is tempting to speculate that the DMs may have arisen from the same l p region. A similar suggestion was made by Atkin and Baker [26]. In h u m a n cervical carcinomas there is often an excess of material from chromosome 1. In one such carcinoma this was not obviously the case but instead DMs were present, the suggestion being that they represented the extra material from chromosome 1. The availability of clones from the TR14 DMs could prove an important tool to test this hypothesis in neuroblastomas. We are particularly grateful to Dr. J. Pritchard for making available the original neuroblastoma biopsy material, and to Dr. L. M. Franks and Bridget T. Hill for their support and encouragement during the course of these studies.
REFERENCES 1. Vogel F (1979): Genetics of retinoblastoma. Hum Genet 52, 1-54. 2. Riccardi VM, Hittner HM, Francke U, Yunis JJ, Ledbetter D, Borges W (1980): The aniridia-Wilms' tumor association: The critical role of chromosome band 11p13. Cancer Genet Cytogenet 2, 131-137. 3. Knudson AG, Strong LC (1972): Mutation and cancer. Neuroblastoma and pleochomocytoma. Am J Hum Genet 24, 514-532. 4. Cowell JK (1982): Double minutes and homogeneously staining regions: Gene amplification in mammalian cells. Ann Rev Genet 16, 21-59. 5. Rupniak HT, Rein G, Powell J, Ryder T, Newman R, Hill BT (1982): Induction of differentiation of a human neuroblastoma cell line by cyclic-AMP. In : Pediatric Oncology; Proc. 13th Meeting Int Soc Pediatr Oncol, C Raybaud, R Clement, G Lebreuil, JL Bernard, eds. Excerpta Medica, Amsterdam, pp 76-80. 6. Cowell JK (1980): Consistent chromosome abnormalities associated with mouse bladder epithelial cell lines transformed in vitro. J Natl Cancer Inst 95,955-961. 7. Miller OJ, Miller DA, Allderdice PW, Dev VG, Grewal MS (1971): Quinacrine fluorescent karyotypes of human diploid and heteroploid cell lines. Cytogenetics 10, 338-346. 8. Brodeur GM, Green AA, Hayes FA, williams KJ, Tsiatis HA (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41, 46784686. 9. Brodeur GM, Sekhon GS, Goldstein MN (1977): Chromosome aberrations in human neuroblastomas. Cancer 40, 2256-2263. 10. Biedler JL, Ross RA, Shanske S, Spengler BA (1980): Human neuroblastoma cytogenetics: Search for significance of homogeneously staining regions and double minute chromosomes. In: Advances in Neuroblastoma Research, AE Evans, ed. Raven Press, New York, pp 81-96.
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11. Haag MM, Soucup SW, Neeley JE (1981): Chromosome analysis of a h u m a n neuroblastoma. Cancer Res 41, 2995-2999. 12. Seeger RC, Rayner SA, Banerjee A, Chang H, Lang WE, Neustein HB, Benedict WF (1977): Morphology, growth, chromosomal pattern and fibrolytic activity of two h u m a n neuroblastoma cell lines. Cancer Res 37, 1364-1371. 13. Biedler JL, Spengler BA (1976): A novel chromosome abnormality in h u m a n neuroblastoma and antifolate resistant Chinese Hamster cell lines in culture. J Natl Cancer Inst 57, 683-695. 14. Balaban-Malenbaum G, Gilbert FW (1977): Double minute chromosomes and the homogeneously staining regions in chromosomes of a h u m a n neuroblastoma cell line. Science 198, 739-741. 15. Gilbert FW, Balaban-Malenbaum G, Breg WR, Gallie B, Ried T, Nichols WW (1981): Hoq mogeneously staining regions in a retinoblastoma cell line. Relevance to tumor induction and progression. J Natl Cancer Inst 67, 301-306. 16. Dolnick BJ, Berenson RJ, Bertino JR, Kaufman RJ, Nunberg JH, Schimke RT (1979): Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining region in L5178Y cells. J Cell Biol 83,394-402. 17. Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA (1978): Amplified dihydrofolate reductase genes are localised to a homogeneously staining region of a single chromosome in a methotrexate resistant Chinese hamster ovary cell line. Proc Natl Acad Sci USA 75, 5553-5556. 18. Wahl G, Vitto L, Padgett RA, Stark GR (1982): Localisation of single copy and amplified CAD genes in Syrian hamster chromosomes using a highly sensitive method for in situ hybridisation. Mol Cell Biol 2,308-319. 19. Sandberg AA, Sakurai M, Holdsworth RN (1972): Chromosomes and the causation of human cancer and leukaemia. VIII. DM chromosomes in a neuroblastoma. Cancer 16, 16711679. 20. Bloch-Shtacher N, Sachs L (1976): Chromosome balance in the control of malignancy. J Cell Physiol 87, 89-100. 21. Yamamoto T, Rabinowitz Z, Sachs L (1973): Identification of the chromosomes that control malignancy. Nature New Biol 234, 247-250. 22. Cowell JK (1979): Chromosome changes associated with epithelial cell transformation with special reference to in vitro systems. In: Neoplastic Transformation in Differentiated Epithelial Cell Systems in Vitro, LM Franks, CB Wigley, eds. Academic Press, London, pp. 259-286. 23. Cowell JK (1981): Chromosome abnormalities associated with salivary gland epithelial cell lines transformed in vitro and in vivo with evidence of a role for genetic imbalance in transformation. Cancer Res 41, 1508-1517. 24. Cowell JK, Wigley CB (1980): Changes in DNA content during in vitro transformation of mouse salivary gland epithelium J Natl Cancer Inst 64, 1443-1449. 25. Cowell JK, Wigley CB (1982): Chromosome changes associated with the progression of cell lines from a preneoplastic to a tumorigenic phenotype during transformation of mouse salivary gland epithelium in vitro. J Natl Cancer Inst 69,425-433. 26. Atkin NB, Baker MC (1980): Cytogenetic observations on a carcinoma of the cervix uteri with double minute chromosome bodies. Eur 1 Cancer 16, 793-797.
ABSTRACT:
The chromosomal analysis of a human neuroblastoma cell line derived from a pediatric patient is presented. The cell line has a modal chromosome number of 64 and contains large numbers of double minutes (DMs). The DMs are stable over many passages in vitro. A consistent feature of the karyotype is an abnormality involving the short arm of chromosome 1, which is consistent with previous reports from human neuroblastomas. The region distal to band lp31 has homogeneously staining characteristics and as such this cell line represents a rare example where DMs and HSRs are constitutive features of the same cells. The possibility that such regions could arise from chromosome rearrangements is discussed.
INTRODUCTION The belief that specific c h r o m o s o m e aberrations m a y be involved in the p r e d i s p o sition to the d e v e l o p m e n t of cancer has been strongly s u p p o r t e d by the findings in several neoplasms of pediatric origin. In two cases the increased probability of tumor d e v e l o p m e n t has been correlated with the inheritance or spontaneous dev e l o p m e n t of small, specific c h r o m o s o m e deletions. The risk of d e v e l o p i n g retinoblastoma has been associated with the deletion of b a n d 13q14 [1] and the deletion of c h r o m o s o m e b a n d 11p13 greatly e n h a n c e d the chance of d e v e l o p i n g Wilms' tumor [2]. Neuroblastomas represent another relatively c o m m o n pediatric cancer w h i c h shows an inherited p r e d i s p o s i t i o n [3]. A c c u m u l a t e d evidence has revealed the association of three specific c h r o m o s o m e abnormalities: aberrations involving the short arm of c h r o m o s o m e 1, double minutes (DMs), and h o m o g e n e o u s l y staining regions (HSRs). Although DMs and HSRs have been reported in tumors from a variety of different cell types, neuroblastomas probably represent the most frequent example [4]. In this report we present the cytogenetic findings from h u m a n neuroblastoma cell line TR14, derived from a pediatric patient, w h i c h contains DMs and an aberration involving the short arm of c h r o m o s o m e 1 w h i c h is possibly an HSR.
From the Department of Haematologyand Oncology,Institute of Child Health, London (J.K.C.)and the Department of Cellular Chemotherapy, Imperial Cancer Research Fund, London. Address requests for reprints to John K. Cowell, Department of Haematology and Oncology, Institute of Child Health, 30 Guilford Street, London WC1, England. Received October 13, 1982; accepted December 1, 1982.
273 © Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 0165-4608/83/$03.00
274
J.K. Cowell et al.
MATERIAL AND METHODS
Cells and Cell Culture The TR14 cell line was derived from biopsy material of a 3-year-old male with stage III disease who had previously been treated with chemotherapy--Vincristine, Cyclophosphamide, Adriamycin, Melphalan (high-dose)--and low-dose radiotherapy. The procedure for isolation of the cell line and the characteristics of the resultant cells have been presented in more detail elsewhere [5]. All ceils were grown in Hams F12 medium supplemented with 10% fetal calf serum and transferred and harvested with trypsin. Chromosome Preparations The cell cycle time of the TR14 cells was approximately 3 days which made it difficult to obtain large numbers of metaphases without excessive treatment with mitotic arresting agents which would cause chromosome contraction. A 2-hr vinblastine treatment proved to be the maximum acceptable, after which the cells were
Figure 1
Representative Giemsa-banded karyotype from the TR14 cell line showing three
copies of the abnormality on chromosome 1.
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harvested and chromosome preparations made using standard air drying techniques [6]. T r y p s i n - G i e s m a b a n d i n g was carried out according to a modified technique described previously [6]. RESULTS In this study 20 well-banded metaphases were analyzed, giving the modal chromosome n u m b e r in TR14 cells as 63 (54%), with a range of 60-64. Sporadic chromosome counts over a 12-month period showed this n u m b e r to be fairly constant during in vitro culture. A representative karyotype of the TR14 cell line is s h o w n in Figure 1. Chromosomal variation among cells could be accounted for by apparently r a n d o m losses and gains of i n d i v i d u a l chromosomes. A n HSR-like region was present on the short arm of chromosome 1 distal to band lp31 (Fig. 2) and the tip had acquired a heavy band. In the majority of cells there were at least two of these markers and sometimes three. Four other chromosome markers were present (Fig. 1), one of w h i c h - - m l - - i n v o l v e s the translocation of the long arm of chromosome 3 onto the long arm of chromosome 1 (Fig. 1) and as such was similar to a marker seen in HeLa cells [7]. The other three markers could not be positively identified. It was possible that the distal region of the chromosome 1 marker was not an HSR but had arisen as a result of chromosome rearrangement (see Discussion). The distal region of l p is normally weakly banded (Fig. 2) and a rearrangement may
Figure 2 (Top) The homogeneously staining region on the short arm of chromosome 1 in TR14. (Bottom) A normal chromosome 1 (left) is compared with the abnormal 1 (right) and illustrates how the homogeneously staining region (HSR) might have arisen from the translocation of an unidentified chromosome region to the tip of the short arm of chromosome 1. A suggested breakpoint is indicated by the arrow.
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J.K. Cowell et al. involve the relocation of an unidentified region to the tip of l p with a breakpoint in lp36. The presence of the distal heavy band suggests that some translocation had occurred although the identity of the material could not be determined.
Double Minutes In the TR14 cell line there were often i n n u m e r a b l e double minutes (Fig. 3, 4) in every cell. T y p i c a l l y they varied in n u m b e r from cell to cell, sometimes c o m p l e t e l y covering the c h r o m o s o m e s (Fig. 3). Karyotypes m a d e 12 months apart i n d i c a t e d that the DMs have persisted in the cultured cells for at least this period of time. Not only was there heterogeneity in DM n u m b e r but size heterogeneity between DMs was also noted in the same cell (Fig. 5). The size of DMs in the TR14 cell line varied from the limits of resolution to relatively large DMs (Fig. 5). The presence of DMs and the HSR-like region in l p was noted in all cells. DISCUSSION The short arm of c h r o m o s o m e 1 is frequently involved in c h r o m o s o m e abnormalities in both neuroblastoma tissue and in cell lines derived from t h e m [8]. Consistent with this general finding, neuroblastoma cell line TR14 also has an aberration involving the short arm of chromosome 1. Deletion of most or part of the short arm has been the most consistent finding [8-11], the deleted region in c o m m o n to t h e m all being that distal to band lp32. In one of these cases this deletion represented the only c h r o m o s o m e abnormality in the cell [9]. In some cases where there was a p p a r e n t l y no deletion of chromosomal material, the short arm of c h r o m o s o m e 1 could be either relocated in the genome [10], or be the recipient site for a translocation or insertion [8]. These findings further i m p l y that disturbance of the genetic material in the short arm of chromosome 1 m a y be important in the d e v e l o p m e n t of neuroblastoma. Many neuroblastomas are n e a r - d i p l o i d [8]; TR14 however was subtetraploid, p r e s u m a b l y as a result of tetraploid formation and c h r o m o s o m e loss, a feature of some other established neuroblastoma cell lines [12]. The presence of two or more copies of the abnormal c h r o m o s o m e 1 suggests that this a b n o r m a l i t y was present in the cell line before endoreduplication, in contrast to the other c h r o m o s o m e markers in TR14 w h i c h were only present in single copy. Other c h r o m o s o m e abnormalities found frequently in neuroblastomas are HSRs [10] where they have been shown to be associated with m a n y different chromosomes. W h e n the HSR was located on c h r o m o s o m e 1 its position was variable, although in one case (IMR 32) it was located w i t h i n the frequently deleted 1p32ter region [11]. In TR14 the aberrant c h r o m o s o m e 1 was cytologically similar to that reported in IMR 32, an i n d e p e n d e n t l y derived cell line [14]. The same HSR w i t h a subterminal pale region and a distally located heavy b a n d was also observed in a retinoblastoma cell line [15]. There is now strong evidence that, in a few specific systems, HSRs represent gene amplification [16-18] and, by analogy, it is generally considered that all other HSRs also represent gene amplification [4]. Cytologically the subterminal region of chromosome 1 in TR14 is characteristic of an HSR but w h e t h e r it has arisen as a result of localized gene amplification or chromosome rearrangement is uncertain. One difficulty in distinguishing between these two possibilities lies in the fact that the l p 3 2 - t e r region is ordinarily faintly banded. Relocation of similar regions "back-to-back" could give the i m p r e s s i o n of an HSR. The presence of a distally located heavy band, not n o r m a l l y present on chromosome 1, further suggests that a rearrangement has occurred at some stage. Such a rearrangement possibly involves
Figure 3 Figure 4
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Giemsa-stained metaphase spread from TR14 showing large numbers of DMs with variable size. A cell from TR14 with fewer but more homogeneously sized DMs (arrows).
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Figure 5 Heterogeneity in DM size within a single cell from TR14. DMs vary from the limits of resolution to a size (arrows) approaching that of the smallest human chromosome.
one of the breakpoints in band l p 3 6 and is thus similar to that in a previous report by Brodeur et al. [9]. It was not possible in this case to determine whether there had been any deletion of chromosomal material during the evolution of the abnormal chromosome. The only detectable short arm material from chromosome 1 was present on the marker chromosome, which possibly makes any large deletion unlikely as this w o u l d represent complete loss of this material from the cell. The constitutive presence of DMs and HSRs in the same cell is rare [4] which is another reason to conclude that the u n b a n d e d region i n TR14 is not an HSR. A n alternative possibility is that two completely different genes have been amplified and in these cells the amplification has been mediated by DM and HSR formation, respectively. The TR14 cell line was not resistant to methotrexate and the identity of the amplified genes is not known. It is also completely u n k n o w n whether the amplified sequences are related to the malignancy of the cells. It may be that there are several different genes present in the DMs. The answers to these questions might be obtained from specific DM-DNA clones from TR14, which w o u l d be a suitable starting material because of the high n u m b e r of DMs per cell and their stability in the cells in vitro. Since neuroblastomas often contain DMs [10] an analysis of this type might prove useful in determining whether the same genes are being amplified in other h u m a n neuroblastomas.
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The frequent presence of DMs and HSRs in h u m a n neuroblastomas suggests that this tumor cell type shows a propensity for the genesis of amplified sequences [19]. Whether these genes are involved in m a l i g n a n c y of the cell is not known. It might be that they represent a genetic imbalance i n the cell responsible for the malignant phenotype. It was suggested by Sachs and co°workers [20, 21] that cancer might be due to chromosome imbalance, defined as a change in the relative dose of certain genes in malignant cells compared with the normal cell. Chromosome imbalance can possibly be generated in m a n y ways [22, 23] i n c l u d i n g deletions and duplications of whole or parts of chromosomes, tetraploid formation and chromosome loss [24, 25], and also from position effect due to chromosome rearrangement [22]. All of these chromosome abnormalities at one time or another have been described associated with chromosome 1 in neuroblastomas [8]. It is possible therefore that imbalance for the 1p32-ter region is important in the genesis of neuroblastoma. Theoretically imbalance can also be produced by specific amplification of the gene(s) relevant to m a l i g n a n c y by the formation of HSRs and DMs. In one particular cell line an HSR has been shown to be located w i t h i n the 1p32-ter region [13]. Since DMs are an alternative form of HSRs [4], it is tempting to speculate that the DMs may have arisen from the same l p region. A similar suggestion was made by Atkin and Baker [26]. In h u m a n cervical carcinomas there is often an excess of material from chromosome 1. In one such carcinoma this was not obviously the case but instead DMs were present, the suggestion being that they represented the extra material from chromosome 1. The availability of clones from the TR14 DMs could prove an important tool to test this hypothesis in neuroblastomas. We are particularly grateful to Dr. J. Pritchard for making available the original neuroblastoma biopsy material, and to Dr. L. M. Franks and Bridget T. Hill for their support and encouragement during the course of these studies.
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