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Cytogenetic analysis in human breast tumors
Cytogenetic Analysis in Human Breast Tumors Sen Pathak
ABSTRACT: Cytogenetic analysis using C-, G-, and Ag-nucleolus organizer region [NOR) staining techniques, performed on established cell lines as well as directly processed breast tumor effusions, revealed that: 1) chromosome No. 1 is involved in translocotion; 2) based on lq translocation chromosome, breast tumors could be classified into two groups; and 3) double minutes and homogeneously staining regions may be present in breast tumor cells in vivo as well as in vitro, and that homogeneously staining regions may exhibit some heterogeneity in staining. The application of chromosome banding techniques to studies of h u m a n tumors has greatly improved our understanding of the role of chromosomes in tumor progression, classifying the tumor based on its genetic constitution, and monitoring of such patients for chemotherapy. The bulk of cytogenetic data has been gathered from the tumors of hemopoietic system. The application of chromosome banding techniques on solid tumors has just begun. The solid tumors are notorious in the sense that it is not always possible to obtain sufficient numbers of suitable metaphase spreads to perform detailed cytogenetic analysis. In direct processing of tumor biopsies the success rate for obtaining suitable mitotic figures is very low. Cytogeneticists have tried to overcome this problem by establishing long-term cell lines from solid tumors and then analyzing their karyotypes. But this practice has a number of flaws: 1) in vitro condition might influence the evolution of a different karyotype that was not present.in vivo, 2) at times normal cells grow faster and take over the tumor cells, and 3) analysis of tumor chromosomes takes a long time because of the delay in establishing the cell line. At M. D Anderson Hospital and Tumor Institute, Dr. Relda Cailleau in the Department of Medicine, has established a number of cell lines from body fluids of breast tumor patients over the last several years [9]. Our initial study of chromosomes using banding techniques was done on some of these established breast tumor cell lines. The modal chromosome numbers in these lines vary from 40 to 59. In a preliminary report we have shown that in seven of these established breast tumor cell lines, the distal part of the long arm of chromosome No. 1 was always involved in translocation [11]. This c o m m o n chromosome abnormality was present in the large majority of metaphases of a given tumor. Since we were looking for a c o m m o n denominator, we did not carefully compare other marker chromosomes present in these cell lines. Therefore, the cell lines are now being analyzed thoroughly and the data will be published as soon as available. From the Department of Biology,Sectionof Cell Biology,The Universityof Texas SystemCancer Center M. D. Anderson Hospital and Tumor Institute, Houston, Texas Address requests for reprints to: Dr. S. Pathak, Department of Biology, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030. Received and accepted September 10, 1979.
© Elsevier North Holland, Inc., 1980 Cancer Genetics and Cytogenetics1,281-289 (1980) 0165-4608/80/01028109502.25
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DIRECT CYTOGENETIC ANALYSIS FROM CELLS OF EFFUSIONS
Because chromosome changes take place rather frequently in vitro, genetic and cytogenetic characteristics obtained from long-term lines may or may not represent the characteristics of the original tumor cells in vivo. For this reason, we decided to examine the effusions from the breast tumor patients directly for chromosome analysis. As in solid tumors, it is not always possible to obtain a good yield of suitable metaphase plates for banding analysis. We process the effusion samples using this protocol: The effusions obtained from the clinics are divided into three containers, one for isozyme analysis, the second for direct chromosome preparations, and the third for establishing long-term cell lines. About 50 ml of the effusion is centrifuged at 1,700 rpm for 5 min. The supernatant is discarded and the pellet is immediately dispersed in a warm (37°C) hypotonic solution for 1 5 - 2 0 min. In our laboratory sodium citrate (0.7%) works well for spreading metaphase chromosomes. We try not to treat our samples with any mitotic arrestants, because then the chromosomes become supercondensed and are good only for counting the number and cannot be used for the induction of Giemsa banding. The cells are fixed in glacial acetic acid and absolute methanol (1:3) fixative and air-dried on acetone-cleaned slides. Some slides are conventionally stained in Giemsa for counting the chromosome number. The remainder of the slides are processed for the induction of chromosome bandings following the routine procedures in this laboratory [32]. INVOLVEMENT OF CHROMOSOME NO. 1 IN BREAST TUMOR
Rowley [36] and later Kakati et al. [17] demonstrated that chromosome No. 1 was frequently involved in rearrangements in certain human tumors. Recently it has been shown that chromosome No. 1 is most frequently involved in marker chromosome formation in a large variety of human tumors, including myeloproliferative diseases, breast, ovarian, lung, malignant melanoma, and colon cancers (see reviews in [18] and [38]}. The first breast tumor cell line (MDA-MB-175) in which a translocation between chromosome Nos. 7 and 1 was observed was from a 56-year-old black woman. The cell line was established from a pleural effusion prior to any chemotherapy. Plates of conventionally stained and a C-banded metaphase from this sample are shown in Figure la and b, respectively. Two large submetacentric markers are shown in Figure la (arrowheads). After C banding, these chromosomes exhibited normal centromeric as well as abnormal intercalary distributions of the constitutive heterochromatin (Fig. lb, arrows). It became evident after G banding that a major part of the lq was tandemly translocated to the long arm of No. 7 [11]. The data on C-banding patterns indicated that a break occurred in the proximal C-band positive segment of lq. However, in other breast tumor lines (13 of them) the break probably took place below the C-band segment in the long arm, because C banding performed on these lines did not show an intercalary heterochromatic segment on lq marker chromosomes. In other breast tumor cell lines lq was translocated to chromosomes 3, 5, 7, 10, 11, and 12 [10]. However, in one of our cell lines (MDA-MB-435), chromosome No. 1 is not involved in translocation. Ayraud et al. [4] have reported lq translocation in five out of seven breast tumor cell lines established from malignant pleural effusions. Out of two human tumor epithelial cell lines from solid breast carcinomas, at least in one, lq translocation is obvious [20]. Most recently, we have been able to demonstrate that lq translocation was present, even in the chromosome preparations made directly from effusions of two breast tumor patients [10,35]. These observations suggest that the lq translocation, although present in other human tumors [18], is not an artifact induced in vitro, but
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Figure 1 Metaphase plates from human breast carcinoma cell line MDA- MB- 175. (a) Conventionally stained metaphase plate with two large submetacentric marker chromosomes (arrowheads). (b) C-banded metaphase plate showing intercalary distribution of constitutive heterochromatin in two marker chromosomes (arrows). See text for the origin of these segments.
that it arose in the tumor cell present in the b o d y of the breast cancer patient. In our samples the marker c h r o m o s o m e involving l q translocation is rarely present in more than one copy. For example, in MDA-MB-468 where the stem line n u m b e r is 35, o n l y one marker involving l q is present. Only in those cell lines where 2S stem line is established, is more than one marker present, except for MDA-MB-175, where 1S n u m b e r is 49 and two marker chromosomes are present (Fig. 1). This is an important
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s. Pathak point because Rowley [37] has emphasized a trisomy of lq in many other human tumors. Thus, it appears that involvement of lq in chromosome rearrangements provides some proliferative advantages for different human tumors. Another structural abnormality in human chromosome No. 1 has been brought to our attention. In a series of papers Atkin and collaborators [1- 3] have shown that in many tumors at least one of the chromosome No. 1 exhibits pericentric inversion or some kind of structural polymorphism in the C-band segment. Such observations led them to suggest that human subjects with pericentric inversion in the C-band segment of chromosome No. 1 might be susceptible to develop tumor. In the majority of our breast tumor cell lines, the C-band polymorphism in at least one homolog of chromosome No. 1 is quite evident.
DOUBLE MINUTE (DM) AND HOMOGENEOUSLY STAINING REGION (HSR) Two unusual and relatively new cytogenetic findings in human tumors are paired, small, DNA-containing spherical bodies called double minutes (Fig. 2) and chromosome segment(s) evenly stained after Giemsa banding known as homogeneously staining regions (Fig. 3). DMs were first described in human lung tumors by Spriggs et ial. [39] and in experimental tumor by Mark [25]. Recently DMs have been described in great detail in neurogenic human tumors and in breast and colon tumor cell lines as well (see [6]). Such bodies are also described in mouse tumors [23, 24]. The number of DMs varies from zero to several hundred in different metaphases of the same tumor. Detailed cytological characterizations of DMs in both human and mouse tumor cell lines have been recently published by Barker and Hsu [6] and Levan and co-workers [23, 24], respectively. As shown in Figure 2, DMs are C-band negative and they do not stain darkly either with G-band or R-band techniques [6]. Another relatively new cytogenetic finding was revealed after Giemsa banding of Chinese hamster cells and human neuroblastoma cell lines which are made resistant to methotrexate [8]. Such chromosome segments are uniformly stained after G-banding treatment, and hence called homogeneously staining regions [7]. Similar HSRs were reported in rat sarcomas and RSV-induced rat tumors [22]. Recently Nielsen [30] and Levan et al. [23] have described HSR-type marker chromosomes or chromosome segment in different laboratory strains of mouse including the SEWA mouse ascites tumors. In direct chromosome preparations from three human mammary carcinomas, HSR-carrying markers were recently demonstrated by Kovacs [19]. An HSRcarrying marker chromosome from the directly processed pleural effusion of a breast tumor patient is shown in Figure 3a. The short arm of this marker has the characteristic G bands of the long arm of No. 7; but the middle segment of its long arm is homogeneously stained. The proximal end of the long arm has two intensely stained Cband segments and the terminal end of the long arm is also C-band positive. This marker chromosome is also present in the established cell line of the patient [35]. Figure 3b shows a short HSR-carrying chromosome from another established breast tumor cell line (MDA-MB-231). HSR of human tumors are mostly C-band negative, as has already been reported by Biedler and Spengler [8]. However, a C-band positive HSR is reported in the karyotypes of two breast tumor samples [19; Peter Barker, personal communication]. The HSR present in the SW-527 (a suspected human breast tumor cell line) does not follow the original definition of HSR proposed by Biedler et al. [7]. As shown in Figure 3c, two markers carrying HSR (arrows) of SW-527 line have banded segments. The proximal one-third segment of the long arm is darkly stained, whereas the rest of the arm is lightly stained. The extreme terminal segments in the long arms have dark and light G bands. Similar HSRs were also observed in human neuroblastoma cells (J. Biedler, personal communication]. Therefore, the history of HSR staining patterns is
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Figure 2 C-banded metaphase spread from a tumor line SW-732. Note the absence of C banding in double minutes. not simple. It could be C-bend positive, negative, and evenly or unevenly stained under G-banding treatment in different cell lines. In four human neuroblastoma cell lines, Balaban-Malenbaum and Gilbert [5] reported that in a metaphase, if DMs were present then, HSR-carrying chromosome was absent. In another metaphase plate of the same sample, if a HSR-carrying marker was present, then DMs were missing. They interpreted this to mean that DMs could be derived from the HSR and vice versa. A similar observation was made by Levan et al. [23] in a mouse tumor cell line. Probably DMs and HSRs are not tumor-specific chromosome abnormalities and their occurrence is not uncommon. As initially speculated by Biedler and Spangler [8] and later experimentally demonstrated by Nunberg et al. [31], HSRs represent specific gene amplification to develop tolerance against certain drug treatments. Presence of HSR in breast tumor samples is not surprising, because these patients are treated with methotrexate in combination with other drugs. NUCLEOLUS ORGANIZER REGION (NOR) IN BREAST TUMORS In the human complement, nucleolus organizer regions are present in the short arms of the acrocentric chromosomes [12]. The NORs can now he readily stained using silver-staining technique [13]. The silver-staining method has clearly shown that the achromatic stalks of the satellites in all D and G group human chromosomes are darkly stained and represent the location of 18 and 28S ribosomal genes [15]. Since D and G group chromosomes are often involved in translocation in both congenital chromosome abnormalities and in human tumor cells, it would be informative to apply the silver-staining technique to identify these regions. Hubbell and Hsu [16] applied the silver-staining technique to nine established cell lines derived from human tumors of various origin. From their figures (Nos. 3b, c, and d) it is apparent that NORs on many chromosomes are displaced from their original locations. Later, Miller and co-workers [26] used this technique on another human tumor cell line. A silver-stained metaphase plate of an established breast tumor
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Figure 3 Giemsa-banded partial metaphase plates from an uncultured breast effusion and two breast tumor cell lines. (a) Metaphase plate from uncultured pleural effusion showing a large marker chromosome with HSR (arrow). (b) Metaphase from MDA-MB-231 showing a small HSR-carrying chromosome (arrow). (c) Appearance of the SW-527 (a suspected breast tumor line) marker chromosomes with G-banded HSR (arrows). cell line is s h o w n in Figure 4. This cell line has a h y p o d i p l o i d stem line n u m b e r of 35. There are only five silver-stained regions (Fig. 4, arrowheads), and two of t h e m are present on b i a r m e d chromosomes. NORs are G-band dull and C-band negative. In a n u m b e r of m a m m a l i a n species the NORs may be e m b e d d e d w i t h i n the C-band positive segments as in laboratory mouse and in the cactus mouse, Peromyscus eremicus. The satellites, w h e n present on h u m a n D a n d G group chromosomes, are C-band positive. The NOR, w h e n located at the terminal end of certain chromosomes, can only be identified by silver staining [13,33,34]. This cytochemical p r o c e d u r e s h o u l d be a p p l i e d on any h u m a n t u m o r or transformed cell lines in order to d e c i p h e r the position of the NOR.
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! Figure 4 Silver-stained metaphase plate from a breast tumor cell line MDA-MB-468, showing only 5 NORs (arrowheads). Two NORs are located on abnormal positions.
INTRASPECIES CELL LINE CONTAMINATION In early 1971, Miller and co-workers applied Q-banding techniques to a number of established human cell lines to identify the marker chromosomes. They identified four marker chromosomes in the HeLa cell line [27]. The HeLa markers, since then, have been extensively used by cytogeneticists to identify intraspecies cell line contamination [14;21] (also see reviews [28] and 29). In many instances such observations might be correct, because cell line contamination is not uncommon in any tissue culture laboratory. However, the presence of several HeLa-like marker chromosomes and 13 identical isozyme loci in a freshly obtained breast tumor pleural effusion and in its established line [35] has changed our concept regarding the absolute utility of marker chromosomes in determining cell line contamination, for it appears that similar rearrangements can be generated repeatedly and independently. From the above discussion, we conclude that: 1) the long arm of chromosome No. 1 is involved in translocation in human breast ~umors and that such translocation is not only present in established cell lines but also in tumor cells in situ; 2) one could use a lq translocation marker to roughly classify breast tumors into two groups; 3) DMs and HSRs may be present in breast tumor cells also; 4) one should use more than one chromosome banding technique to identify modified chromosomes; and 5) more samples should be studied cytogenetically in order to interpret the data to aid tumor diagnosis, application of chemotherapy, and evaluation of cancer risk.
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s. Pathak The author is indebted to Col. A. Leibovitz and Dr. R. Cailleau for cell lines, Dr. K. L. SatyaPrakash for Figure 4, and Dr. T. C. Hsu for going through the first draft. This work was supported in part by a Research Grant VC-21 from the American Cancer Society and a joint research project with the John S. Dunn Research Foundation, Houston, Texas.
REFERENCES 1. Atkin NB (1977): Chromosome 1 heteromorphism in patients with malignant disease: a constitutional marker for a high risk group? Br Med J 1,358. 2. Atkin NB, Baker MC (1977): Pericentric inversion of chromosome 1: frequency and possible association with cancer. Cytogenet Cell Genet 19, 180-184. 3. Atkin NB, Pickthall VJ (1977): Chromosomes 1 in 14 ovarian cancers heterochromatin variants and structural changes. Hum Genet 38, 25 - 33. 4. Ayraud N, Lambert JC, Tribollet KH, Basteris B (1977): Etude cytog~netique comparative de sept carcinomes dSrigine mammaire. Ann Genet 20, 171 - 177. 5. Balaban-Malenbaum G, Gilbert F (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. 6. Barker PE, Hsu TC (1979): Double minutes in h u m a n carcinoma cell lines, with special reference to breast tumors. J Natl Cancer Inst 62,257 - 262. 7. Biedler JL, Albrecht AM, Spengler BA (1974) Non-banding homogeneous chromosome regions in cells with very high dihydrofolate reductase levels. Genetics (Suppl) 77, 4 - 5. 8. Biedler JL, Spengler BA (1976): Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science 191,185 -187. 9. Cailleau R, Olive, M, Cruciger QVJ (1978): Long-term h u m a n breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro 14, 911 -915. 10. Cruciger Q, Pathak S, Barker P, Cailleau R, Eckles N, Blumenschein G (1978): Consistent translocation of l q chromosome in metastatic breast cancer tissue and cell l i n e s - c l i n i c a l correlations. AFCR Oncol (abstr) 433 A. 11. Cruciger QVJ, Pathak S, Cailleau, R. (1976): Human breast carcinomas: marker chromosomes involving l q in seven cases. Cytogenet Cell Genet 17, 231-235. 12. Ferguson-Smith MA (1964): The sites of nucleolus formation in h u m a n pachytene chromosomes. Cytogenetics 3,124 - 134. 13. Goodpasture C, Bloom SE (1975): Visualization of nucleolus organizer regions in mammalian chromosomes using silver stain. Chromosoma 53, 3 7 - 50. 14. Heneen WK (1976): HeLa cells and their possible contamination of other cell lines: karyotype studies. Hereditas 8 2 , 2 1 7 - 2 4 8 . 15. Hsu TC, Spirito SE, Pardue ML (1975): Distribution of 18 ÷ 28S ribosomal genes in mammalian genomes. Chromosoma 53, 25 - 36. 16. Hubbell HR, Hsu TC (1977): Identification of nucleolus organizer regions (NORs) in normal and neoplastic h u m a n cells by the silver-staining technique. Cytogenet Cell Genet 19, 185-196. 17. Kakati S, Hayata I, Oshimura M, Sandberg AA (1975): Chromosomes and causation of human cancer and leukemia X handing patterns in cancerous effusions. Cancer 36, 1729-1738. 18. Kovacs G (1976): Abnormalities of chromosome No. 1 in human solid malignant tumors. Int J Cancer 2 1 , 6 8 8 - 6 9 4 . 19. Kovacs G (1979): Homogeneously staining regions on marker chromosomes in malignancy. Int J Cancer 23,299 - 301. 20. Lasfarques EY, Coutinho WG, Redfield ES (1978): Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst 6 1 , 9 6 7 - 9 7 8 21. Lavappa KS, Macy ML, Shannon JE (1976): Examination of ATCC stocks for HeLa marker chromosomes in human cell lines. Nature 259, 2 1 1 - 213. 22. Levan A, Levan G, Mitelman F (1977): Chromosomes and cancer. Hereditas 86, 15 - 30.
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23. Levan A, Levan G, Mandahl N (1978): A new chromosome type replacing the double minutes in a mouse tumor. Cytogenet Cell Genet 20, 12 - 23. 24. Levan G, Mandahl N, Bregula U, Klein G, Levan A (1976): Double minute chromosomes are not centromeric regions of the host chromosomes. Hareditas 83, 83 -90. 25. Mark J (1967): Double-minutes. A chromosomal aberration in Rous sarcomas in mice. Hereditas 57, 1 - 22. 26. Miller DA, Dev VG, Tantravahi R, Croce CM, Miller OJ (1978): Human tumor and rodenth u m a n hybrid cells with an increased number of active h u m a n NORs. Cytogenet Cell Genet 21, 3 3 - 4 1 . 27. Miller OJ, Miller DA, Allderdice PW, Dev VG, Grewal MS (1971): Quinacrine fluorescent karyotypes of h u m a n diploid and heteroploid cell lines. Cytogenetics 10,338 - 346. 28. Nelson-Rees WA (1978): The identification and monitoring of cell line specificity. In: Origin and Natural History of Cell Lines. Alan R. Liss, Inc., New York, pp. 2 5 - 79. 29. Nelson-Rees WA, Flandarmeyer, RR, Hawthorne PK (1974): Banded marker chromosomes as indicators of intraspecies cellular contamination. Science 184, 1093 - 1096. 30. Nielsen K (1976): Chromosomal evolution in the Ehrlich-Lettr6 complex of hyperdiploid mouse ascites tumours: results from seven laboratory strains. Hereditas 84, 77-107. 31. Nunberg JH, Kaufman RJ, Schimke RT, Urlaub G, Chasin LA (1978): Amplified dihydrofolate reductase genes are localized 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. 32. Pathak S (1976): Chromosome banding techniques. J Reprod Med 17, 25 - 28. 33. Pathak S (1979): Cytogenetic research techniques in humans and laboratory animals that can be applied most profitably to livestock. J Dairy Sci 6 2 , 8 3 6 - 843. 34. Pathak S, Kieffer NM (1979): Sterility in hybrid cattle I. Distribution of constitutive heterochromatin and nucleolus organizer regions in somatic and meiotic chromosomes. Cytogenet Cell Genet 24, 4 2 - 52. 35. Pathak S, Siciliano MJ, Cailleau R, Wiseman CL, Hsu TC (1979): A h u m a n breast adenocarcinoma with chromosome and isoenzyme markers similar to those of the HeLa line. J Natl Cancer Inst 62,263 -271. 36. Rowley JD [1975): Nonrandom chromosomal abnormalities in hematologic disorders of man. Proc Natl Acad Sci USA 72, 152 -156. 37. Rowley JD (1977): Mapping of h u m a n chromosomal regions related to neoplasia: evidence from chromosomes 1 and 17. Proc Natl Acad Sci USA 74, 5729-5733. 38. Rowley JD (1978): Abnormalities of chromosome No. 1: significance in malignant transformation. Virchows Arch B Cell Patho129, 139 - 144. 39. Spriggs AI, Boddington MM, Clarke CM (1962): Chromosomes of h u m a n cancer cells. Br MedJ 2, 1431-1435.
ABSTRACT: Cytogenetic analysis using C-, G-, and Ag-nucleolus organizer region [NOR) staining techniques, performed on established cell lines as well as directly processed breast tumor effusions, revealed that: 1) chromosome No. 1 is involved in translocotion; 2) based on lq translocation chromosome, breast tumors could be classified into two groups; and 3) double minutes and homogeneously staining regions may be present in breast tumor cells in vivo as well as in vitro, and that homogeneously staining regions may exhibit some heterogeneity in staining. The application of chromosome banding techniques to studies of h u m a n tumors has greatly improved our understanding of the role of chromosomes in tumor progression, classifying the tumor based on its genetic constitution, and monitoring of such patients for chemotherapy. The bulk of cytogenetic data has been gathered from the tumors of hemopoietic system. The application of chromosome banding techniques on solid tumors has just begun. The solid tumors are notorious in the sense that it is not always possible to obtain sufficient numbers of suitable metaphase spreads to perform detailed cytogenetic analysis. In direct processing of tumor biopsies the success rate for obtaining suitable mitotic figures is very low. Cytogeneticists have tried to overcome this problem by establishing long-term cell lines from solid tumors and then analyzing their karyotypes. But this practice has a number of flaws: 1) in vitro condition might influence the evolution of a different karyotype that was not present.in vivo, 2) at times normal cells grow faster and take over the tumor cells, and 3) analysis of tumor chromosomes takes a long time because of the delay in establishing the cell line. At M. D Anderson Hospital and Tumor Institute, Dr. Relda Cailleau in the Department of Medicine, has established a number of cell lines from body fluids of breast tumor patients over the last several years [9]. Our initial study of chromosomes using banding techniques was done on some of these established breast tumor cell lines. The modal chromosome numbers in these lines vary from 40 to 59. In a preliminary report we have shown that in seven of these established breast tumor cell lines, the distal part of the long arm of chromosome No. 1 was always involved in translocation [11]. This c o m m o n chromosome abnormality was present in the large majority of metaphases of a given tumor. Since we were looking for a c o m m o n denominator, we did not carefully compare other marker chromosomes present in these cell lines. Therefore, the cell lines are now being analyzed thoroughly and the data will be published as soon as available. From the Department of Biology,Sectionof Cell Biology,The Universityof Texas SystemCancer Center M. D. Anderson Hospital and Tumor Institute, Houston, Texas Address requests for reprints to: Dr. S. Pathak, Department of Biology, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030. Received and accepted September 10, 1979.
© Elsevier North Holland, Inc., 1980 Cancer Genetics and Cytogenetics1,281-289 (1980) 0165-4608/80/01028109502.25
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DIRECT CYTOGENETIC ANALYSIS FROM CELLS OF EFFUSIONS
Because chromosome changes take place rather frequently in vitro, genetic and cytogenetic characteristics obtained from long-term lines may or may not represent the characteristics of the original tumor cells in vivo. For this reason, we decided to examine the effusions from the breast tumor patients directly for chromosome analysis. As in solid tumors, it is not always possible to obtain a good yield of suitable metaphase plates for banding analysis. We process the effusion samples using this protocol: The effusions obtained from the clinics are divided into three containers, one for isozyme analysis, the second for direct chromosome preparations, and the third for establishing long-term cell lines. About 50 ml of the effusion is centrifuged at 1,700 rpm for 5 min. The supernatant is discarded and the pellet is immediately dispersed in a warm (37°C) hypotonic solution for 1 5 - 2 0 min. In our laboratory sodium citrate (0.7%) works well for spreading metaphase chromosomes. We try not to treat our samples with any mitotic arrestants, because then the chromosomes become supercondensed and are good only for counting the number and cannot be used for the induction of Giemsa banding. The cells are fixed in glacial acetic acid and absolute methanol (1:3) fixative and air-dried on acetone-cleaned slides. Some slides are conventionally stained in Giemsa for counting the chromosome number. The remainder of the slides are processed for the induction of chromosome bandings following the routine procedures in this laboratory [32]. INVOLVEMENT OF CHROMOSOME NO. 1 IN BREAST TUMOR
Rowley [36] and later Kakati et al. [17] demonstrated that chromosome No. 1 was frequently involved in rearrangements in certain human tumors. Recently it has been shown that chromosome No. 1 is most frequently involved in marker chromosome formation in a large variety of human tumors, including myeloproliferative diseases, breast, ovarian, lung, malignant melanoma, and colon cancers (see reviews in [18] and [38]}. The first breast tumor cell line (MDA-MB-175) in which a translocation between chromosome Nos. 7 and 1 was observed was from a 56-year-old black woman. The cell line was established from a pleural effusion prior to any chemotherapy. Plates of conventionally stained and a C-banded metaphase from this sample are shown in Figure la and b, respectively. Two large submetacentric markers are shown in Figure la (arrowheads). After C banding, these chromosomes exhibited normal centromeric as well as abnormal intercalary distributions of the constitutive heterochromatin (Fig. lb, arrows). It became evident after G banding that a major part of the lq was tandemly translocated to the long arm of No. 7 [11]. The data on C-banding patterns indicated that a break occurred in the proximal C-band positive segment of lq. However, in other breast tumor lines (13 of them) the break probably took place below the C-band segment in the long arm, because C banding performed on these lines did not show an intercalary heterochromatic segment on lq marker chromosomes. In other breast tumor cell lines lq was translocated to chromosomes 3, 5, 7, 10, 11, and 12 [10]. However, in one of our cell lines (MDA-MB-435), chromosome No. 1 is not involved in translocation. Ayraud et al. [4] have reported lq translocation in five out of seven breast tumor cell lines established from malignant pleural effusions. Out of two human tumor epithelial cell lines from solid breast carcinomas, at least in one, lq translocation is obvious [20]. Most recently, we have been able to demonstrate that lq translocation was present, even in the chromosome preparations made directly from effusions of two breast tumor patients [10,35]. These observations suggest that the lq translocation, although present in other human tumors [18], is not an artifact induced in vitro, but
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Figure 1 Metaphase plates from human breast carcinoma cell line MDA- MB- 175. (a) Conventionally stained metaphase plate with two large submetacentric marker chromosomes (arrowheads). (b) C-banded metaphase plate showing intercalary distribution of constitutive heterochromatin in two marker chromosomes (arrows). See text for the origin of these segments.
that it arose in the tumor cell present in the b o d y of the breast cancer patient. In our samples the marker c h r o m o s o m e involving l q translocation is rarely present in more than one copy. For example, in MDA-MB-468 where the stem line n u m b e r is 35, o n l y one marker involving l q is present. Only in those cell lines where 2S stem line is established, is more than one marker present, except for MDA-MB-175, where 1S n u m b e r is 49 and two marker chromosomes are present (Fig. 1). This is an important
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s. Pathak point because Rowley [37] has emphasized a trisomy of lq in many other human tumors. Thus, it appears that involvement of lq in chromosome rearrangements provides some proliferative advantages for different human tumors. Another structural abnormality in human chromosome No. 1 has been brought to our attention. In a series of papers Atkin and collaborators [1- 3] have shown that in many tumors at least one of the chromosome No. 1 exhibits pericentric inversion or some kind of structural polymorphism in the C-band segment. Such observations led them to suggest that human subjects with pericentric inversion in the C-band segment of chromosome No. 1 might be susceptible to develop tumor. In the majority of our breast tumor cell lines, the C-band polymorphism in at least one homolog of chromosome No. 1 is quite evident.
DOUBLE MINUTE (DM) AND HOMOGENEOUSLY STAINING REGION (HSR) Two unusual and relatively new cytogenetic findings in human tumors are paired, small, DNA-containing spherical bodies called double minutes (Fig. 2) and chromosome segment(s) evenly stained after Giemsa banding known as homogeneously staining regions (Fig. 3). DMs were first described in human lung tumors by Spriggs et ial. [39] and in experimental tumor by Mark [25]. Recently DMs have been described in great detail in neurogenic human tumors and in breast and colon tumor cell lines as well (see [6]). Such bodies are also described in mouse tumors [23, 24]. The number of DMs varies from zero to several hundred in different metaphases of the same tumor. Detailed cytological characterizations of DMs in both human and mouse tumor cell lines have been recently published by Barker and Hsu [6] and Levan and co-workers [23, 24], respectively. As shown in Figure 2, DMs are C-band negative and they do not stain darkly either with G-band or R-band techniques [6]. Another relatively new cytogenetic finding was revealed after Giemsa banding of Chinese hamster cells and human neuroblastoma cell lines which are made resistant to methotrexate [8]. Such chromosome segments are uniformly stained after G-banding treatment, and hence called homogeneously staining regions [7]. Similar HSRs were reported in rat sarcomas and RSV-induced rat tumors [22]. Recently Nielsen [30] and Levan et al. [23] have described HSR-type marker chromosomes or chromosome segment in different laboratory strains of mouse including the SEWA mouse ascites tumors. In direct chromosome preparations from three human mammary carcinomas, HSR-carrying markers were recently demonstrated by Kovacs [19]. An HSRcarrying marker chromosome from the directly processed pleural effusion of a breast tumor patient is shown in Figure 3a. The short arm of this marker has the characteristic G bands of the long arm of No. 7; but the middle segment of its long arm is homogeneously stained. The proximal end of the long arm has two intensely stained Cband segments and the terminal end of the long arm is also C-band positive. This marker chromosome is also present in the established cell line of the patient [35]. Figure 3b shows a short HSR-carrying chromosome from another established breast tumor cell line (MDA-MB-231). HSR of human tumors are mostly C-band negative, as has already been reported by Biedler and Spengler [8]. However, a C-band positive HSR is reported in the karyotypes of two breast tumor samples [19; Peter Barker, personal communication]. The HSR present in the SW-527 (a suspected human breast tumor cell line) does not follow the original definition of HSR proposed by Biedler et al. [7]. As shown in Figure 3c, two markers carrying HSR (arrows) of SW-527 line have banded segments. The proximal one-third segment of the long arm is darkly stained, whereas the rest of the arm is lightly stained. The extreme terminal segments in the long arms have dark and light G bands. Similar HSRs were also observed in human neuroblastoma cells (J. Biedler, personal communication]. Therefore, the history of HSR staining patterns is
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Figure 2 C-banded metaphase spread from a tumor line SW-732. Note the absence of C banding in double minutes. not simple. It could be C-bend positive, negative, and evenly or unevenly stained under G-banding treatment in different cell lines. In four human neuroblastoma cell lines, Balaban-Malenbaum and Gilbert [5] reported that in a metaphase, if DMs were present then, HSR-carrying chromosome was absent. In another metaphase plate of the same sample, if a HSR-carrying marker was present, then DMs were missing. They interpreted this to mean that DMs could be derived from the HSR and vice versa. A similar observation was made by Levan et al. [23] in a mouse tumor cell line. Probably DMs and HSRs are not tumor-specific chromosome abnormalities and their occurrence is not uncommon. As initially speculated by Biedler and Spangler [8] and later experimentally demonstrated by Nunberg et al. [31], HSRs represent specific gene amplification to develop tolerance against certain drug treatments. Presence of HSR in breast tumor samples is not surprising, because these patients are treated with methotrexate in combination with other drugs. NUCLEOLUS ORGANIZER REGION (NOR) IN BREAST TUMORS In the human complement, nucleolus organizer regions are present in the short arms of the acrocentric chromosomes [12]. The NORs can now he readily stained using silver-staining technique [13]. The silver-staining method has clearly shown that the achromatic stalks of the satellites in all D and G group human chromosomes are darkly stained and represent the location of 18 and 28S ribosomal genes [15]. Since D and G group chromosomes are often involved in translocation in both congenital chromosome abnormalities and in human tumor cells, it would be informative to apply the silver-staining technique to identify these regions. Hubbell and Hsu [16] applied the silver-staining technique to nine established cell lines derived from human tumors of various origin. From their figures (Nos. 3b, c, and d) it is apparent that NORs on many chromosomes are displaced from their original locations. Later, Miller and co-workers [26] used this technique on another human tumor cell line. A silver-stained metaphase plate of an established breast tumor
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(b)
(c)
Figure 3 Giemsa-banded partial metaphase plates from an uncultured breast effusion and two breast tumor cell lines. (a) Metaphase plate from uncultured pleural effusion showing a large marker chromosome with HSR (arrow). (b) Metaphase from MDA-MB-231 showing a small HSR-carrying chromosome (arrow). (c) Appearance of the SW-527 (a suspected breast tumor line) marker chromosomes with G-banded HSR (arrows). cell line is s h o w n in Figure 4. This cell line has a h y p o d i p l o i d stem line n u m b e r of 35. There are only five silver-stained regions (Fig. 4, arrowheads), and two of t h e m are present on b i a r m e d chromosomes. NORs are G-band dull and C-band negative. In a n u m b e r of m a m m a l i a n species the NORs may be e m b e d d e d w i t h i n the C-band positive segments as in laboratory mouse and in the cactus mouse, Peromyscus eremicus. The satellites, w h e n present on h u m a n D a n d G group chromosomes, are C-band positive. The NOR, w h e n located at the terminal end of certain chromosomes, can only be identified by silver staining [13,33,34]. This cytochemical p r o c e d u r e s h o u l d be a p p l i e d on any h u m a n t u m o r or transformed cell lines in order to d e c i p h e r the position of the NOR.
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! Figure 4 Silver-stained metaphase plate from a breast tumor cell line MDA-MB-468, showing only 5 NORs (arrowheads). Two NORs are located on abnormal positions.
INTRASPECIES CELL LINE CONTAMINATION In early 1971, Miller and co-workers applied Q-banding techniques to a number of established human cell lines to identify the marker chromosomes. They identified four marker chromosomes in the HeLa cell line [27]. The HeLa markers, since then, have been extensively used by cytogeneticists to identify intraspecies cell line contamination [14;21] (also see reviews [28] and 29). In many instances such observations might be correct, because cell line contamination is not uncommon in any tissue culture laboratory. However, the presence of several HeLa-like marker chromosomes and 13 identical isozyme loci in a freshly obtained breast tumor pleural effusion and in its established line [35] has changed our concept regarding the absolute utility of marker chromosomes in determining cell line contamination, for it appears that similar rearrangements can be generated repeatedly and independently. From the above discussion, we conclude that: 1) the long arm of chromosome No. 1 is involved in translocation in human breast ~umors and that such translocation is not only present in established cell lines but also in tumor cells in situ; 2) one could use a lq translocation marker to roughly classify breast tumors into two groups; 3) DMs and HSRs may be present in breast tumor cells also; 4) one should use more than one chromosome banding technique to identify modified chromosomes; and 5) more samples should be studied cytogenetically in order to interpret the data to aid tumor diagnosis, application of chemotherapy, and evaluation of cancer risk.
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s. Pathak The author is indebted to Col. A. Leibovitz and Dr. R. Cailleau for cell lines, Dr. K. L. SatyaPrakash for Figure 4, and Dr. T. C. Hsu for going through the first draft. This work was supported in part by a Research Grant VC-21 from the American Cancer Society and a joint research project with the John S. Dunn Research Foundation, Houston, Texas.
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