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Cytogenetic analysis of two C-1300 murine neuroblastoma cell lines expressing discordant malignant behavior
Cytogenetic Analysis of Two C-1300 Murine Neuroblastoma Cell Lines Expressing Discordant Malignant Behavior Jeffrey Sawyer and Mendel Tuchman
ABSTRACT: Cytogenetic analysis of two murine neuroblastoma cell lines that show different malignant expression revealed consistent differences in the chromosomal composition between the two lines. The MNB-T1 (high-malignancy) cell line showed increased madal chromosome number, presence of homogeneously staining regions (HSRs), double minutes (drain) and evidence of secondary chromosomal events when compared with the MNB-T2 (low-malignancy) cell line, which did not express these cytogenetic characteristics. Several of the cytogenetic events that have occurred in these murine cell lines, such as HSRs, dmin, and chromosomal deletions, have also been reported in human neuroblastoma cell lines. When comparisons are made for the involvement of chromosomal regions known to be involved in human neuroblastoma, similar tumor specific regions seem to be involved in the homologous mouse chromosomes.
INTRODUCTION Human neuroblastoma tumors and cell lines contain chromosomal rearrangements preferentially involving the short arm of chromosome 1 [1]. In addition, evidence of gene amplification has been found to be expressed at the cytogenetic level by the presence of homogeneously staining regions (HSRs) and extrachromosomal genomic material known as double minutes (drain). The characterization of these cytogenetic events has helped to elucidate the clonal evolution and tumor progression in human neuroblastoma and has also provided prognostic information [2]. The use of the mouse as a model for human disease and genetic mapping purposes is widespread. Studies of genetic events in the mouse, such as chromosome rearrangements, are important because the mouse is the most extensively mapped mammalian species. For example, in the analysis of murine plasmacytoma cell lines, chromosomal translocations associated with cellular protooncogene activation have been found by chromosomal banding, thus providing information that can be used as a model for human Burkitt lymphoma [3]. The investigation of the cytogenetic events that characterize mouse neuroblastoma cell lines should provide insight into these same types of chromosomal events and also provide information as to the use of the mouse neuroblastoma as a model for this human disease.
From the Department of Pathology, University of Arkansas for Medical Sciences, and Cytogenetics Laboratory, Arkansas Children's Hospital, Little Rock, Arkansas (J. S.) and the Departments of Pediatrics and Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota (M. T.).
Address reprint requests to: Jeffrey Sawyer, Cytogenetics Laboratory, Arkansas Children's Hospital, 800 Marshall Street, Little Rock, AR 72202. Received February 8, 1989; accepted April 18, 1989.
99 © 1989 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 42:99-106 (1989) 0165-4608/89/$03.50
100
J. Sawyer and M. Tuchman We have recently reported that the enzymatic activity of pyrimidine degradation inversely correlates with the neoplastic expression of two different C-1300 murine neuroblastoma cell lines [4, 5]. The cells of the high-malignancy line (MNB-T1) are round, show poor attachment to the surface of the culture flask, and are uniformly lethal when injected back into A/J mice; on the other hand, the low-malignancy cells (MNB-T2) are flat, attach well to surfaces, and cause death only in 20% of the inoculated animals [4]. Cells from either line show very few short neurite formations. Cells of new cultures established from in situ tumors showed the characteristics of T1 cells for several passages. T2 cells eventually appeared in the culture and became the predominant cells if the culture was maintained for many passages (estimated 50100 passages). We have not observed morphologic or enzymatic conversion of T2predominant cultures back to T1. It is currently unknown to us whether the T2 cells represent a subclone of cells within the MNB tumor that are better adapted to growth in culture or whether they are the result of positive selection of mutant cells during culture. Observations made during dilution cloning of T1 and T2 cells indicated that clones of T1 cells converted to cells with T2 characteristics, and therefore favor the latter hypothesis. Similar descriptions of two neuroblastoma cell types in culture have been reported in mice and human tumors and are referred to as "neuroblast like" or "N type" (similar to our MNB-T1) cells and "epithelial like" or "S type" (similar to MNB-T2) cells [6-9]. Chromosomal banding analysis of the T1 and T2 mouse neuroblastoma cell lines will demonstrate the cytogenetic relationship between the cell lines and also reveal their individual chromosomal characteristics. This study should also help in the understanding of the chromosome changes associated with tumor progression in mouse neuroblastoma cells.
MATERIALS AND METHODS Cells were maintained in culture as described in detail elsewhere [4]. The harvesting of the cells was carried out as follows. The cells were treated with a dilute Colcemid solution (4 ~g/ml) for a period of 2 hours. The cells were centrifuged at 100 g for 10 minutes and then resuspended in a hypotonic solution of 500 0.075 M KC1 and 50% 0.8% sodium citrate at 37°C for 10 minutes. The cells were then centrifuged and fixed with 5 : 2 methanol : acetic acid. Chromosome preparations were banded by treatment with I N HC1 for 20 minutes followed by a 90-minute incubation in 50% formamide and 2 × SSC. The slides were then rinsed in tap water and dehydrated in 95% ethanol for 30 minutes. Following dehydration and drying at room temperature for 2 hours, the slides were stained with a 3 : 1 (v/v) phosphate buffer : Wright stain mix.
RESULTS The MNB-T1 and MNB-T2 cell lines are very similar in many cytogenetic features, although they vary in chromosome number and have slightly different complex chromosomal rearrangements. Two hundred cells from both cell lines were studied for chromosome counts and the presence of extra chromosomal material such as HSRs and dmin. The T1 cell line has a modal chromosome number of 62, with a range of 58-63. The T2 cell line has a modal chromosome number of 60, with a range of 59-61. In general, most cells are near-triploid, having three copies of each chromosome, although certain chromosomes are represented by as many as four or five copies. In the T1 cell line, the frequency of putative HSRs were rare events (about 3%), and the frequency of dmin was 4% (Fig. 1), whereas, no evidence of HSRs or drain was observed in the T2 cell line.
Cytogenetics of Mouse Neuroblastoma Cells
101
B
Figure 1
Metaphase spread of a MWB-T1 cell showing presence of dmin (arrows).
Both m u r i n e neuroblastoma cell lines have two normal copies of c h r o m o s o m e 1, and a third c h r o m o s o m e 1 deleted at band h l (Figs. 2 and 3). The cell lines have two normal copies of c h r o m o s o m e 2, and one isochromosome 2, w h i c h is involved in a c o m p l e x translocation of u n k n o w n origin. One normal chromosome 3 and one Robertsonian translocation of c h r o m o s o m e 3 are present in both cell lines. O n l y two copies of c h r o m o s o m e 4 are present in both cell lines, w h i c h may be significant in the r e l a t i o n s h i p to the h u m a n neuroblastoma m o d e l (see Discussion) (Figs. 2 and 3). There are three copies of chromosome 5 in the T1 and four in the T2 cell line. Four n o r m a l copies of c h r o m o s o m e 6 are present in the T1 cell line, w h i l e in the T2 cell line three normal and one a p p a r e n t l y deleted # 6 are present. Two copies of chromosome 7, and three copies of chromosome 8 are found in both lines, although two of the three copies of c h r o m o s o m e 8 are translocated in a Robertsonian fashion onto the X c h r o m o s o m e (Figs. 2 and 3). One normal chromosome 9 and one deleted # 9 is found in each line. Three copies of c h r o m o s o m e 10 are present, one as an isochromosome in both cell lines. Three copies of chromosome 11 are found in both lines, two with different quantities of extra material translocated onto their distal ends. Two normal copies of c h r o m o s o m e 12 and generally one chromosome 13 are found in both cell lines. Three copies of chromosome 14 and four copies of c h r o m o s o m e 15 are found consistently in both T1 and T2 (Figs. 2 and 3). Two normal copies of c h r o m o s o m e 16 are present and one c h r o m o s o m e 16 has a translocation onto its distal e n d in both lines. Three normal copies of c h r o m o s o m e 17 are present consistently in both lines. One normal copy each of chromosomes 18 and 19 are found in the T1 line, whereas in the T2 cell line one normal c h r o m o s o m e 18 and two normal copies of c h r o m o s o m e 19 are generally found. Several small marker c h r o m o s o m e s are present in both cell lines but are too small to be identified with metaphase
102
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banding techniques (Figs. 2 and 3). Individual cells sometimes had extra or missing chromosomes of certain types but generally followed the above descriptions.
Specific Chromosomal Changes The major cytogenetic differences between the two cell lines involve the presence of dmin, HSRs, and the translocation of relatively small chromosome segments in the T1 cell line, which do not occur in the T2 cell line. The isochromosome 2 has undergone translocations on both distal segments, with the original isochromosome remaining stable up to band D1 in both arms. The difference between this chromosome in lines T1 and T2 is in the short arm, where material has been translocated in the T2 cell line (Fig. 4A). The isochromosome 3, present in both cell lines, differs by having additional material translocated onto one of the arms in the T2 cell line (Fig. 4B). The isochromosome 10 has apparently developed what appears to be an achromatic region in one arm (Fig. 4C), with a translocation of unknown origin present.
103
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Figure 3 Karyotype of a MNB-T2 cell showing near triploid chromosome constitution. M indicates marker chromosomes. The achromatic region was not observed in the T1 cell line. This may be a secondary chromosomal event, resulting from tissue culture artifact. The presence of drain in the T1 cell line may be related to an interesting finding in this cell line. An apparent amplification event (HSR) on the distal end of the X chromosome (Fig. 4D) was observed in several cells. The finding of a putative HSR on the distal end of the t(X;8) chromosome and the finding of dmin-like structures in this same region may indicate some type of relationship between HSRs and dmin (Fig. 4D) [10]. This same chromosome appears normal in most cells, most likely indicating that if this is an amplification event, it occurs only rarely. DISCUSSION Specific chromosomal rearrangements are a common finding in many cancers such as leukemias, lymphomas, and solid tumors. Specific chromosomal findings have been shown to correlate with the prognosis in certain diseases and have been impli-
104
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Figure 4 Selected pairs of chromosomes showing consistent chromosomal differences between T1 and T2 cell lines. A. Chromosomes 2 showing distal translocation (brackets) seen in T2 cell line and not in T1. B. Chromosomes 3 showing translocation (brackets] on distal end of isochromosome in T2 cell line not seen in T1. C. Isochromosomes 10 showing achromatic region (open arrow) in T2 cells not seen in T1. D. Translocation X;8 chromosome from T1 cell line showing putative HSR (bracket) and apparent drain-like structures (arrows] not seen in T2 cell line. Dotted line indicates position of centromeres.
cated in the step-wise progression of ceils to a more malignant p h e n o t y p e [11]. The best e x a m p l e of this p h e n o m e n o n is the consistent chromosomal rearrangement in chronic m y e l o g e n o u s leukemia, t(9;22). The occurrence of additional changes in the karyotype of i n d i v i d u a l s with this leukemia usually indicates a poor prognosis. In h u m a n neuroblastoma, the significant chromosomal event has been the deletion or rearrangement of the short arm of c h r o m o s o m e I [14]. The additional finding of drain seems to lead to a poorer prognosis [2]. Cytogenetic studies indicate that the cell lines T1 and T2 exhibit karyotypic constancy over most of the chromosomes and that the normal c h r o m o s o m e groups r e m a i n e d similar. The most striking variation in the two lines occurs in the n u m b e r of small marker chromosomes, All cells in both cell lines showed structural abnormalities consisting of translocations and deletions, but drain and HSRs were found
Cytogenetics of Mouse Neuroblastoma Cells
105
only in the T1 cell line. These findings probably reflect the instability of the genome in these malignant cells. The more malignant T1 cells show a higher n u m b e r of marker c h r o m o s o m e s (deletions and translocations) than the less malignant T2 cells. This is consistent with findings in other murine tumor cell lines [12]. In the mouse, the region that is homologous to the h u m a n c h r o m o s o m e l p is the c h r o m o s o m e 4 [13]. It m a y be significant that in both of these cell lines, only two copies of c h r o m o s o m e 4 are present, one fewer than the near-triploid n u m b e r of three c h r o m o s o m e s represented in most of the karyotype. The absence of mouse chromosome 4 m a y be equivalent to the l p deletions or translocations seen in h u m a n neuroblastomas. W i t h the apparent absence or at least rearrangement of one copy of c h r o m o s o m e 4, it w o u l d appear that these cell lines at the chromosomal level m a y have types of c h r o m o s o m a l events analogous to the h u m a n neuroblastoma model. Of particular interest is the finding that in mouse neuroblastoma, as in the human, the presence of d m i n and HSRs seem to be related to amplified gene sequences associated w i t h cellular oncogenes. These findings i m p l y that oncogene amplification plays a role in the d e v e l o p m e n t of neuroblastoma, possibly acting through the prod u c t i o n of excess product. Even though gene amplification m a y not be one of the first steps by w h i c h cells become cancerous, it most likely contributes to the progression of cancers to a more malignant form. The different c h r o m o s o m a l changes observed in the two cell lines probably reflect the difference in the malignant expression of the cells w h e n injected back into the mouse [4]. If is difficult to speculate w h i c h of the numerous specific c h r o m o s o m a l changes m a y be involved in the p h e n o t y p i c differences noted in these two cell lines, but the presence of the HSRs and d m i n in the T1 cell line can probably be c o n s i d e r e d the most i m p o r t a n t difference. REFERENCES 1. Brodeur G, Green A, Hayes A, Williams K, Tsiatis A (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4878-4685. 2. Brodeur G, Seegar R, Schwab M, Varmus H, Bishop J (1986): Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224:1121-1124. 3. Klein G (1981): The role of gene dosage and genetic position in carcinogenesis. Nature 294:313-318. 4. Tuchman M, O'Dea RF, Ramnaraine MLR, Mirkin BL (1988): Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors. J Clin Invest 81:425430. 5. Williams CS, Tuchman M (1989): Correlations of dihydropyrimidine dehydrogenase, thymidine phosphorylase and thymidine kinase activities in strongly and weakly malignant murine neuroblastoma cells. Int J Cancer 43:901-904. 6. Lanks KW, Lonbardo JM (1981): Clonal variation in cultured neuroblastoma cells. II. The relationship of increased intracellular cyclic AMP content to increased anchorage requirement for growth and flattened morphology. J Cell Physiol 190:45-51. 7. Ross RA, Spengler BA, Biedler JL (1983): Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst 71:741-749. 8. Komada Y, Azuma E, Kamiya H, Sakurai M (1986): Phenotypic profile of human neuroblastoma cell lines: Association with morphological characteristics. Br J Cancer 54:711-715. 9. Bernal S, Thompson R, Gilbert F, Baylin SB (1983): In vitro and in vivo growth characteristics of two different cell populations in an established line of human neuroblastoma. Cancer Res 43:1256-1260. 10. Brookwell R, Hunt F (1988): Formation of double minutes by breakdown of a homogeneously staining region in a refractory anemia with excess blasts. Cancer Genet Cytogenet 34:47-52. 11. Yunis JJ (1983): The chromosomal basis of human neoplasia. Science 221:227-236.
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12. Dzarlieva R, Schirrmacher V, Fusenig N (1982): Cytogenetic changes during tumor progression towards invasion, metastasis and immune escape in the Eb/ESb model system. Int J Cancer 30:633-642. 13. Sawyer J, Hozier J (1986): High resolution of mouse chromosomes: Banding conservation between man and mouse. Science 232:1632-1635. 14. Yasuhide H, Naotoshi K, Toshiya I, Ryoji H, Noboru N, Hiromu M, Yamamoto K (1989): Cytogenetic findings and prognosis in neuroblastoma with emphasis on marker chromosome 1. Cancer 63:126-132.
ABSTRACT: Cytogenetic analysis of two murine neuroblastoma cell lines that show different malignant expression revealed consistent differences in the chromosomal composition between the two lines. The MNB-T1 (high-malignancy) cell line showed increased madal chromosome number, presence of homogeneously staining regions (HSRs), double minutes (drain) and evidence of secondary chromosomal events when compared with the MNB-T2 (low-malignancy) cell line, which did not express these cytogenetic characteristics. Several of the cytogenetic events that have occurred in these murine cell lines, such as HSRs, dmin, and chromosomal deletions, have also been reported in human neuroblastoma cell lines. When comparisons are made for the involvement of chromosomal regions known to be involved in human neuroblastoma, similar tumor specific regions seem to be involved in the homologous mouse chromosomes.
INTRODUCTION Human neuroblastoma tumors and cell lines contain chromosomal rearrangements preferentially involving the short arm of chromosome 1 [1]. In addition, evidence of gene amplification has been found to be expressed at the cytogenetic level by the presence of homogeneously staining regions (HSRs) and extrachromosomal genomic material known as double minutes (drain). The characterization of these cytogenetic events has helped to elucidate the clonal evolution and tumor progression in human neuroblastoma and has also provided prognostic information [2]. The use of the mouse as a model for human disease and genetic mapping purposes is widespread. Studies of genetic events in the mouse, such as chromosome rearrangements, are important because the mouse is the most extensively mapped mammalian species. For example, in the analysis of murine plasmacytoma cell lines, chromosomal translocations associated with cellular protooncogene activation have been found by chromosomal banding, thus providing information that can be used as a model for human Burkitt lymphoma [3]. The investigation of the cytogenetic events that characterize mouse neuroblastoma cell lines should provide insight into these same types of chromosomal events and also provide information as to the use of the mouse neuroblastoma as a model for this human disease.
From the Department of Pathology, University of Arkansas for Medical Sciences, and Cytogenetics Laboratory, Arkansas Children's Hospital, Little Rock, Arkansas (J. S.) and the Departments of Pediatrics and Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota (M. T.).
Address reprint requests to: Jeffrey Sawyer, Cytogenetics Laboratory, Arkansas Children's Hospital, 800 Marshall Street, Little Rock, AR 72202. Received February 8, 1989; accepted April 18, 1989.
99 © 1989 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 42:99-106 (1989) 0165-4608/89/$03.50
100
J. Sawyer and M. Tuchman We have recently reported that the enzymatic activity of pyrimidine degradation inversely correlates with the neoplastic expression of two different C-1300 murine neuroblastoma cell lines [4, 5]. The cells of the high-malignancy line (MNB-T1) are round, show poor attachment to the surface of the culture flask, and are uniformly lethal when injected back into A/J mice; on the other hand, the low-malignancy cells (MNB-T2) are flat, attach well to surfaces, and cause death only in 20% of the inoculated animals [4]. Cells from either line show very few short neurite formations. Cells of new cultures established from in situ tumors showed the characteristics of T1 cells for several passages. T2 cells eventually appeared in the culture and became the predominant cells if the culture was maintained for many passages (estimated 50100 passages). We have not observed morphologic or enzymatic conversion of T2predominant cultures back to T1. It is currently unknown to us whether the T2 cells represent a subclone of cells within the MNB tumor that are better adapted to growth in culture or whether they are the result of positive selection of mutant cells during culture. Observations made during dilution cloning of T1 and T2 cells indicated that clones of T1 cells converted to cells with T2 characteristics, and therefore favor the latter hypothesis. Similar descriptions of two neuroblastoma cell types in culture have been reported in mice and human tumors and are referred to as "neuroblast like" or "N type" (similar to our MNB-T1) cells and "epithelial like" or "S type" (similar to MNB-T2) cells [6-9]. Chromosomal banding analysis of the T1 and T2 mouse neuroblastoma cell lines will demonstrate the cytogenetic relationship between the cell lines and also reveal their individual chromosomal characteristics. This study should also help in the understanding of the chromosome changes associated with tumor progression in mouse neuroblastoma cells.
MATERIALS AND METHODS Cells were maintained in culture as described in detail elsewhere [4]. The harvesting of the cells was carried out as follows. The cells were treated with a dilute Colcemid solution (4 ~g/ml) for a period of 2 hours. The cells were centrifuged at 100 g for 10 minutes and then resuspended in a hypotonic solution of 500 0.075 M KC1 and 50% 0.8% sodium citrate at 37°C for 10 minutes. The cells were then centrifuged and fixed with 5 : 2 methanol : acetic acid. Chromosome preparations were banded by treatment with I N HC1 for 20 minutes followed by a 90-minute incubation in 50% formamide and 2 × SSC. The slides were then rinsed in tap water and dehydrated in 95% ethanol for 30 minutes. Following dehydration and drying at room temperature for 2 hours, the slides were stained with a 3 : 1 (v/v) phosphate buffer : Wright stain mix.
RESULTS The MNB-T1 and MNB-T2 cell lines are very similar in many cytogenetic features, although they vary in chromosome number and have slightly different complex chromosomal rearrangements. Two hundred cells from both cell lines were studied for chromosome counts and the presence of extra chromosomal material such as HSRs and dmin. The T1 cell line has a modal chromosome number of 62, with a range of 58-63. The T2 cell line has a modal chromosome number of 60, with a range of 59-61. In general, most cells are near-triploid, having three copies of each chromosome, although certain chromosomes are represented by as many as four or five copies. In the T1 cell line, the frequency of putative HSRs were rare events (about 3%), and the frequency of dmin was 4% (Fig. 1), whereas, no evidence of HSRs or drain was observed in the T2 cell line.
Cytogenetics of Mouse Neuroblastoma Cells
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B
Figure 1
Metaphase spread of a MWB-T1 cell showing presence of dmin (arrows).
Both m u r i n e neuroblastoma cell lines have two normal copies of c h r o m o s o m e 1, and a third c h r o m o s o m e 1 deleted at band h l (Figs. 2 and 3). The cell lines have two normal copies of c h r o m o s o m e 2, and one isochromosome 2, w h i c h is involved in a c o m p l e x translocation of u n k n o w n origin. One normal chromosome 3 and one Robertsonian translocation of c h r o m o s o m e 3 are present in both cell lines. O n l y two copies of c h r o m o s o m e 4 are present in both cell lines, w h i c h may be significant in the r e l a t i o n s h i p to the h u m a n neuroblastoma m o d e l (see Discussion) (Figs. 2 and 3). There are three copies of chromosome 5 in the T1 and four in the T2 cell line. Four n o r m a l copies of c h r o m o s o m e 6 are present in the T1 cell line, w h i l e in the T2 cell line three normal and one a p p a r e n t l y deleted # 6 are present. Two copies of chromosome 7, and three copies of chromosome 8 are found in both lines, although two of the three copies of c h r o m o s o m e 8 are translocated in a Robertsonian fashion onto the X c h r o m o s o m e (Figs. 2 and 3). One normal chromosome 9 and one deleted # 9 is found in each line. Three copies of c h r o m o s o m e 10 are present, one as an isochromosome in both cell lines. Three copies of chromosome 11 are found in both lines, two with different quantities of extra material translocated onto their distal ends. Two normal copies of c h r o m o s o m e 12 and generally one chromosome 13 are found in both cell lines. Three copies of chromosome 14 and four copies of c h r o m o s o m e 15 are found consistently in both T1 and T2 (Figs. 2 and 3). Two normal copies of c h r o m o s o m e 16 are present and one c h r o m o s o m e 16 has a translocation onto its distal e n d in both lines. Three normal copies of c h r o m o s o m e 17 are present consistently in both lines. One normal copy each of chromosomes 18 and 19 are found in the T1 line, whereas in the T2 cell line one normal c h r o m o s o m e 18 and two normal copies of c h r o m o s o m e 19 are generally found. Several small marker c h r o m o s o m e s are present in both cell lines but are too small to be identified with metaphase
102
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2 Karyotype of a MNB-T1 ceil showing near-~ip|oid chromosome constitution. M indicates marker chromosomes.
banding techniques (Figs. 2 and 3). Individual cells sometimes had extra or missing chromosomes of certain types but generally followed the above descriptions.
Specific Chromosomal Changes The major cytogenetic differences between the two cell lines involve the presence of dmin, HSRs, and the translocation of relatively small chromosome segments in the T1 cell line, which do not occur in the T2 cell line. The isochromosome 2 has undergone translocations on both distal segments, with the original isochromosome remaining stable up to band D1 in both arms. The difference between this chromosome in lines T1 and T2 is in the short arm, where material has been translocated in the T2 cell line (Fig. 4A). The isochromosome 3, present in both cell lines, differs by having additional material translocated onto one of the arms in the T2 cell line (Fig. 4B). The isochromosome 10 has apparently developed what appears to be an achromatic region in one arm (Fig. 4C), with a translocation of unknown origin present.
103
Cytogenetics of Mouse Neuroblastoma Cells
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Figure 3 Karyotype of a MNB-T2 cell showing near triploid chromosome constitution. M indicates marker chromosomes. The achromatic region was not observed in the T1 cell line. This may be a secondary chromosomal event, resulting from tissue culture artifact. The presence of drain in the T1 cell line may be related to an interesting finding in this cell line. An apparent amplification event (HSR) on the distal end of the X chromosome (Fig. 4D) was observed in several cells. The finding of a putative HSR on the distal end of the t(X;8) chromosome and the finding of dmin-like structures in this same region may indicate some type of relationship between HSRs and dmin (Fig. 4D) [10]. This same chromosome appears normal in most cells, most likely indicating that if this is an amplification event, it occurs only rarely. DISCUSSION Specific chromosomal rearrangements are a common finding in many cancers such as leukemias, lymphomas, and solid tumors. Specific chromosomal findings have been shown to correlate with the prognosis in certain diseases and have been impli-
104
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Figure 4 Selected pairs of chromosomes showing consistent chromosomal differences between T1 and T2 cell lines. A. Chromosomes 2 showing distal translocation (brackets) seen in T2 cell line and not in T1. B. Chromosomes 3 showing translocation (brackets] on distal end of isochromosome in T2 cell line not seen in T1. C. Isochromosomes 10 showing achromatic region (open arrow) in T2 cells not seen in T1. D. Translocation X;8 chromosome from T1 cell line showing putative HSR (bracket) and apparent drain-like structures (arrows] not seen in T2 cell line. Dotted line indicates position of centromeres.
cated in the step-wise progression of ceils to a more malignant p h e n o t y p e [11]. The best e x a m p l e of this p h e n o m e n o n is the consistent chromosomal rearrangement in chronic m y e l o g e n o u s leukemia, t(9;22). The occurrence of additional changes in the karyotype of i n d i v i d u a l s with this leukemia usually indicates a poor prognosis. In h u m a n neuroblastoma, the significant chromosomal event has been the deletion or rearrangement of the short arm of c h r o m o s o m e I [14]. The additional finding of drain seems to lead to a poorer prognosis [2]. Cytogenetic studies indicate that the cell lines T1 and T2 exhibit karyotypic constancy over most of the chromosomes and that the normal c h r o m o s o m e groups r e m a i n e d similar. The most striking variation in the two lines occurs in the n u m b e r of small marker chromosomes, All cells in both cell lines showed structural abnormalities consisting of translocations and deletions, but drain and HSRs were found
Cytogenetics of Mouse Neuroblastoma Cells
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only in the T1 cell line. These findings probably reflect the instability of the genome in these malignant cells. The more malignant T1 cells show a higher n u m b e r of marker c h r o m o s o m e s (deletions and translocations) than the less malignant T2 cells. This is consistent with findings in other murine tumor cell lines [12]. In the mouse, the region that is homologous to the h u m a n c h r o m o s o m e l p is the c h r o m o s o m e 4 [13]. It m a y be significant that in both of these cell lines, only two copies of c h r o m o s o m e 4 are present, one fewer than the near-triploid n u m b e r of three c h r o m o s o m e s represented in most of the karyotype. The absence of mouse chromosome 4 m a y be equivalent to the l p deletions or translocations seen in h u m a n neuroblastomas. W i t h the apparent absence or at least rearrangement of one copy of c h r o m o s o m e 4, it w o u l d appear that these cell lines at the chromosomal level m a y have types of c h r o m o s o m a l events analogous to the h u m a n neuroblastoma model. Of particular interest is the finding that in mouse neuroblastoma, as in the human, the presence of d m i n and HSRs seem to be related to amplified gene sequences associated w i t h cellular oncogenes. These findings i m p l y that oncogene amplification plays a role in the d e v e l o p m e n t of neuroblastoma, possibly acting through the prod u c t i o n of excess product. Even though gene amplification m a y not be one of the first steps by w h i c h cells become cancerous, it most likely contributes to the progression of cancers to a more malignant form. The different c h r o m o s o m a l changes observed in the two cell lines probably reflect the difference in the malignant expression of the cells w h e n injected back into the mouse [4]. If is difficult to speculate w h i c h of the numerous specific c h r o m o s o m a l changes m a y be involved in the p h e n o t y p i c differences noted in these two cell lines, but the presence of the HSRs and d m i n in the T1 cell line can probably be c o n s i d e r e d the most i m p o r t a n t difference. REFERENCES 1. Brodeur G, Green A, Hayes A, Williams K, Tsiatis A (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4878-4685. 2. Brodeur G, Seegar R, Schwab M, Varmus H, Bishop J (1986): Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224:1121-1124. 3. Klein G (1981): The role of gene dosage and genetic position in carcinogenesis. Nature 294:313-318. 4. Tuchman M, O'Dea RF, Ramnaraine MLR, Mirkin BL (1988): Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors. J Clin Invest 81:425430. 5. Williams CS, Tuchman M (1989): Correlations of dihydropyrimidine dehydrogenase, thymidine phosphorylase and thymidine kinase activities in strongly and weakly malignant murine neuroblastoma cells. Int J Cancer 43:901-904. 6. Lanks KW, Lonbardo JM (1981): Clonal variation in cultured neuroblastoma cells. II. The relationship of increased intracellular cyclic AMP content to increased anchorage requirement for growth and flattened morphology. J Cell Physiol 190:45-51. 7. Ross RA, Spengler BA, Biedler JL (1983): Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst 71:741-749. 8. Komada Y, Azuma E, Kamiya H, Sakurai M (1986): Phenotypic profile of human neuroblastoma cell lines: Association with morphological characteristics. Br J Cancer 54:711-715. 9. Bernal S, Thompson R, Gilbert F, Baylin SB (1983): In vitro and in vivo growth characteristics of two different cell populations in an established line of human neuroblastoma. Cancer Res 43:1256-1260. 10. Brookwell R, Hunt F (1988): Formation of double minutes by breakdown of a homogeneously staining region in a refractory anemia with excess blasts. Cancer Genet Cytogenet 34:47-52. 11. Yunis JJ (1983): The chromosomal basis of human neoplasia. Science 221:227-236.
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12. Dzarlieva R, Schirrmacher V, Fusenig N (1982): Cytogenetic changes during tumor progression towards invasion, metastasis and immune escape in the Eb/ESb model system. Int J Cancer 30:633-642. 13. Sawyer J, Hozier J (1986): High resolution of mouse chromosomes: Banding conservation between man and mouse. Science 232:1632-1635. 14. Yasuhide H, Naotoshi K, Toshiya I, Ryoji H, Noboru N, Hiromu M, Yamamoto K (1989): Cytogenetic findings and prognosis in neuroblastoma with emphasis on marker chromosome 1. Cancer 63:126-132.