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_ipr*-T"^*â„¢-r\= [CANCER RESEARCH 33, 3259-3265, December 1973J Distribution of Chromosome Constitutive Heterochromatin of Syrian Hamster Cells Transformed by Chemical Carcinogens J. A. DiPaolo1 and N. C. Popescu2 Cytogenetics and Cytology Section, Biology Branch, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 20014 SUMMARY The utilization of the C band technique for constitutive heterochromatin has permitted further analysis of both nor mal and marker chromosomes that were found following neoplastic transformation and in tumors derived from the transformed cells. Syrian hamster cells that have been transformed by chemical carcinogens have chromosomes with constitutive heterochromatin characteristic of the species. In addition to the completely heterochromatic short arms of the submetacentric autosomes, the long arms of both X chromosomes and E20 are entirely heterochro matic. Heterochromatin alterations occurred in only 2 dif ferent tumor cultures and in no transformed lines and thus are not a causative factor in transformation. The C band technique facilitated the analysis of marker chromosomes. INTRODUCTION When a number of neoplastic transformed lines, each of which had been obtained in vitro, and several of the corre sponding tumors obtained from injecting the transformed cells into animals were analyzed by reproducible Giemsa (G) or quinacrine (Q) chromosomal banding techniques, 10 markers as well as the individual chromosomes belong ing to the different chromosome pairs of the Syrian ham ster karyotype were identifiable (8). It was possible to determine the origin of the marker chromosomes and to conclude that in malignant transformation of the chromo somal changes, including changes in number of chromo somes as verified by the banding patterns, are reflections of secondary alterations rather than being causally related to the cancer. Procedures now exist for differentiating constitutive heterochromatin from euchromatin areas (2). Chromo somes with similar horizontal banding patterns may differ in their heterochromatin distribution (C pattern). Hetero chromatin is a complex subject and the concepts concern ing it are filled with contradictions (5, 19, 21). However, it is generally accepted that heterochromatin represents blocks or regions on chromosomes that are tightly con'To whom reprint requests should be addressed, at National Cancer Institute, Building 37, Room 2A13, Bethesda, Md. 200I4. 2Visiting Associate. National Cancer Institute. NIH. Permanent ad dress: Oncological Institute, Bucharest, Romania. Received August 23, 1973; accepted September 18, 1973. densed, genetically inactive, and in terms of DNA syn thesis late replicating. Variations in the amount of constitutive heterochroma tin per cell have been considered responsible for changes in phenotypic effects referred to as position effects (20). Chro mosomal aberrations commonly observed in neoplasia could involve heterochromatin because of its known sus ceptibility to breakage by mutagens (14). The direct expo sure of Syrian or Chinese hamster cells to polycyclic hydrocarbons has resulted in wide distribution of carcino gen-associated breaks in different chromosome regions (3, 13). Although the response to carcinogen may be coin cidental with areas known to be rich in heterochromatin, one result accompanying chromosome breakage is lethality related to concentration of carcinogen (J. A. DiPaolo and N. C. Popescu, unpublished data). This study presents observations of heterochromatin distribution of chromo somes of 9 transformed cell lines and 4 primary tumors (obtained by injecting the transformed cells) all of which have been studied in terms of horizontal bands. MATERIALS AND METHODS Heterochromatin of metaphase chromosomes was local ized by a modification of the technique of Arrighi and Hsu (2). Chromosomes of 9 in vitro transformed cell lines were examined at the 7 13 passage whereas the 4 tumor cul tures were examined at the 1st or 2nd passage subsequent to removal of the tumor from the animal. Chromosome DNA replication pattern was studied by using thymidine-3H incorporation and autoradiography. Monolayer cultures in log phase were incubated with thymidine-3H (6.7 ci/mM, 1.0 juCi/ml) for 1 hr. The medium containing thymidine-3H was replaced with fresh medium containing 0.04 ng colcemid per ml of medium 2.5 hr before the cultures were harvested and chromosomes were pre pared. For studies of early replication patterns, the cul tures were incubated with thymidine-3H for 30 min followed by treatment with cold thymidine (100-fold concentration) 9 hr before fixation of the cells. Metaphase spreads were subjected to G chromosome banding technique. After the metaphases were stained and photographed, the stain was removed with absolute methanol and cells rinsed with deionized water. Autoradiography was performed with NTB2 Kodak emulsion for 6 days at 4°.After being de veloped and fixed, the slides were conventionally stained with 5% Giemsa in distilled water. The metaphases were DECEMBER 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 3259 J. A. DiPaolo and N. C. Popescu relocated and the adequately labeled ones were photo graphed. regions of the tumor cells. The study of early and late DNA replication (Fig. 2, a and c) shows that the heterochromatic portion of the marker has a sequence of replication similar to that of the sex chromosomes. In addition, the short arm RESULTS of Chromosome A4 which is a normally late labeling is free of label at the end of the S phase (Fig. 2c). A similar situa Of 9 different transformed cell lines obtained from treat tion was associated with the new heterochromatic translo ment with BP3, aflatoxin B,, PS, or 4NQO and 4 tumors cation to the B9 chromosome. The origin of marker examined, abnormal heterochromatin segments were found chromosomes by ordinary staining procedures is difficult, in only 2 tumors derived from cell lines transformed by if not impossible (Fig. 2, b and d). BP and 4NQO. Karyotypes of BP45t that had been stained Because of the finding after viral (7) or chemical (16) with quinacrine mustard showed, in addition to the ex transformation of extra chromosomes that appeared to pected banding pattern, a diffuse, fluorescent, translocated mimic E20, additional documentation concerning M3 is segment to the short arm of 1 of the B9 chromosomes (Fig. presented. In Fig. 3, Pair E20 and the marker chromo la). The occurrence of this characteristic is approximately some M 3 are compared by conventional staining, DNA 30% and consequently is not considered a marker in the replication, and C band procedure. The confusion of M3 same sense as are M, and M 3 which were found in more with E20, which occurs in conventional staining technique, than 50% of the metaphases examined. In 4NQO16t (Fig. is resolved by studying the DNA replication pattern or C 16), a marker chromosome, Ma, consisted of a nonbanded, bands. During late replication the long arms of both E20 darkly stained translocation to the short arm of the A4 chromosomes are heavily labeled but M3 is not labeled. In chromosome. This marker can be contrasted to Marker terms of C bands it has been shown previously (Fig. lc) that Mb which has a banded translocation to the long arm of in tumor cells the long arms of E20 are heterochromatic but C14. With the C band technique, normal heterochromatin that M 3 has heterochromatic regions associated with the distribution was found in all 4NQO16t chromosomes iden dual centromeres. tified as normal by G band technique. Markers M6 and M7 (Fig. 3) were associated with the The Syrian hamster heterochromatin chromosome pat PS-transformed cell line and tumors; both have subtetratern differs from most species in that all the submetacentric autosomes have short arms that are totally heterochro- ploid modes (8). The C banding procedures provide addi tional information about markers M6and M,. M 6 involves matic. In addition, the long arm of both X chromosomes a translocation to the heterochromatic arm of the X chro as well as the long arm of the smallest metacentric are en mosome. After the addition of the translocation to the X tirely heterochromatic (Fig. \c). Both female X chromo to form M6, the basic X continues to exhibit the banding somes exhibit identical G (18) and C band patterns. Further pattern determined with the G technique and is late in repli analysis of chromosomes of this metaphase (Fig. \c) yields cating for its entire length, consistent with the concept that additional information concerning chromosome markers the inert X chromosome is involved. Although cells of the that have been previously identified by the techniques for PS-transformed line and tumor possess 4 X chromosomes, horizontal bands (G, Q, or trypsin-Giemsa). Because of the 2 of which are late replicating along the entire length, only uncommon C pattern, it is possible again to detect M,, 1 chromosome possesses a translocation. The translocation M 2, and M3. The heterochromatic pattern of M i is identi does not alter the C band distribution of the X chromo cal to that of an A2, thus confirming that the translocation some. This is in contrast to the addition of heterochromatic was euchromatic and autosomal in origin. It was concluded regions reported above for BP45t and 4NQO161. M7 is a that the origin of M2 was a result of a centric fusion of a new metacentric marker chromosome with the character D16 and D17 (8). The C band technique proves that only 1 istic banding pattern of the long arms of Chromosomes A1 centromere is present. By ordinary staining techniques, M3 and A2, neither of which are late replicating. In contrast to could be confused with Pair E20. The utilization of the C the metacentric M3, which possessed a double centromere, band technique shows that, whereas Pair E20 (arrows) has M 7has retained only 1centromere. darkly stained long arms, the M3 has only centrally located heterochromatin. In a metaphase spread from a cell of the 4NQO tumor (Fig. \d), C band preparation shows again DISCUSSION that one-half of each X consists of darkly stained hetero chromatin. The only deviation in heterochromatic pattern Examination of cell lines for heterochromatin has been occurs in the chromosome marker, Ma. This marker oc limited to chromosomes of a kidney line from an adult curred as a result of a translocation to the short arm of A4. African monkey (4), BALB/3T3 cells (17), and an aneuA similar type of translocation was found in Tumor BP45t; ploid line derived from Chinese hamster ovary (6). In however, in that case the translocation occurred to the short BALB/3T3 cells transformed by direct application of 7,12-dimethylbenz(a)anthracene or /V-methyl-/V'-nitro-./Varm of a B9 chromosome (Fig. \a). Chromosome replication in the presence of thymidine-3H nitrosoguanidine, the heterochromatin pattern was similar provides additional information of these heterochromatic to the control BALB/3T3 line (17). The formation of tu mors in BALB/c mice provided evidence that the cells 3The abbreviations used are: BP, benzo(a)pyrene; PS, propanesultone; were neoplastic (9). In the current study, the chromosomes 4NQO, 4-nitroquinoline /V-oxide. of Syrian hamster transformed cell lines and tumors 3260 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Chromosome Constitutive Heterochromatin that have been previously examined for horizontal bands were examined for constitutive heterochromatin (8). All transformed lines had normal constitutive heterochromatin distribution similar to that described previously by Hsu and Arrighi (12) and late DNA replication pattern as reported by Gallon and Holt (10). Also both sex chromosomes of the female Syrian hamster had identical G band patterns (18); the current study shows that one-half of each X chromo some is totally heterochromatic. With thymidine-3H and autoradiographic technique, only one of the X chromo somes is late labeling and becomes completely inactive in a random fashion as suggested by Lyon (15). In 2 of the tumors studied, BP45t and 4NQO161, hetero chromatic segments were translocated to the short arm of Chromosomes B9 and A4, respectively. Such rearrange ments can be considered of secondary significance repre senting another type of chromosomal alteration responsible for in vivo tumor progression. In both cases the translocated segments have a sequence of late replication similar to the sex chromosomes. One effect of the heterochromatic translocation to the short arm of B9 or A4 was an alteration of the replication time of the original short arms which were no longer late replicating. The alteration in the total amount of heterochromatin in the genome may be responsible for a type of position effect resulting in activation of normally repressed chromosome segments. The origin of this new heterochromatic region is unknown since the source of such large segments would normally be the sex chromosomes; however, in all metaphases and karyotypes, both X's are present and normal. The most likely possibility is that the translocated segments were autosomal and euchromatic and underwent a process of inactivation and condensation. In another case, there was a translocation to only 1 X chromosome of the tetraploid PS66-transformed cell line and tumor. This translocation was not heterochromatic material but was attached to the totally inert X chromo some. No change occurred in either the replication time or the heterochromatic pattern of the involved X chromo some. In a recent study of chromatin from minimal-devia tion rat hepatomas, it was concluded that on the basis of template activity and electrophoretic profiles the major nonhistone proteins compared with normal liver chromatin (1). This suggests that some of the difference between the chromatin of hepatomas and liver may be responsible for malignant progression rather than reflective of the process of neoplastic transformation. Transformation by a variety of chemical carcinogens ap pears to occur without any changes in the heterochromatin distribution of Syrian hamster cells. Although chromosome breaks have been observed in cells following exposure to a variety of chemical carcinogens with toxic properties (Refs. 3 and 13; J. A. DiPaolo and N. C. Popescu, unpub lished data), the constitutive heterochromatin has not been studied. Changes in constitutive heterochromatin of human cells produced by toxic concentrations of mitomycin have been reported to result in lesions in heterochromatin areas in 1 of 10 cultures (11). Although alkylating carcinogens may produce the same type of alteration, the likelihood of their relevance to neoplastic transformation is probably DECEMBER minimal since no alteration in heterochromatin was ob served with such chemicals. C band analysis provided information concerning the centromeric areas of the chromosomes as well as confirmatory evidence of some markers that otherwise may be confused since they mimic other chromosomes. For example, on the basis of C band and late DNA replication, the origin of M3 was definitely established. M3 has a distinct replication sequence pattern, C band stain, and 2 centromeres all consistent with the conclusion that M3 is a result of the fu sion of the short arms from C15 and F21. Without this analysis, the origin of M3 might be confused because it has certain limited similarities to a B9 that has been deleted below the centromere on the long arm. In M2 and M7, which consist of the long arms of different chromosomes, the markers retained only 1 centromere. Thus, C band analysis furnished additional information concerning 3 of 10 markers first recognized by horizontal band analysis. REFERENCES 1. Arnold. A. E., Buksas, M. M., and Young, K. E. Comparative Study of Some Properties of Chromatin from Two "Minimal Deviation" Hepatomas. Cancer Res., 33: 1169-1176, 1973. 2. Arrighi, F. E., and Hsu, T. C. Localization of Heterochromatin in Hu man Chromosomes. Cytogenetics, 10: 81-86, 1971. 3. Benedict, W. F. Early Changes in Chromosomal Number and Struc ture after Treatment of Fetal Hamster Cultures with Transforming Doses of Polycyclic Hydrocarbons. J. Nati. Cancer Inst., 49: 585 590. 1972. 4. Bianchi, N. O., and Ayres, J. Heterochromatin Location on Chro mosomes of Normal and Transformed Cells from African Green Monkey (Cercopithecus aethiops). Exptl. Cell Res., 68: 253-258, 1971. 5. Comings, D. E. The Structure and Function of Chromatin. Advan. Human Genet., 3: 237-431, 1972. 6. Deaven, L. L., and Petersen, D. F. The Chromosomes of CHO, an Aneuploid Chinese Hamster Cell Line: G-Band, C-Band, and Auto radiographic Analyses. Chromosoma, 41: 129 144, 1973. 7. Defendi, V., and Lehman, J. M. Transformation of Hamster Cells in Vitro by Polyoma Virus: Morphological, Karyological, Immunological and Transplantation Characteristics. J. Cellular Comp. Physiol.,6<5:351 409, 1965. 8. DiPaolo, J. A., Popescu, N. C., and Nelson, R. L. Chromosomal Banding Patterns and in Vitro Transformation of Syrian Hamster Cells. Cancer Res., 33: 3250-3258, 1973. 9. DiPaolo, J. A., Takano, K., and Popescu, N. C. Quantitation of Chemically Induced Neoplastic Transformation of BALB/3T3 Cloned Cell Lines. Cancer Res., 32: 2686-2695, 1972. 10. Gallon, M., and Holt, S. DNA Replication Patterns of the Sex Chro mosomes in Somatic Cells of the Syrian Hamster. Cytogenetics, 3: 97-111, 1964. 11. Hoehn, H., and Martin, G. M. Heritable Alteration of Human Con stitutive Heterochromatin Induced by Mitomycin C. Exptl. Cell Res., 75: 275-278, 1972. 12. Hsu, T., and Arrighi, F. Distribution of Constitutive Heterochromatin in Mammalian Chromosomes. Chromosoma, 34: 243 253, 1971. 13. Kato, R. Chromosome Breakage Induced by a Carcinogenic Hydro carbon in Chinese Hamster Cells and Human Leukocytes in Vitro. Hereditas, 59: 120 141, 1968. 14. Kihlman, B. A. Molecular Mechanisms of Chromosome Breakage 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 3261 J. A. Di Paolo and N. C. Popescu and Rejoining. Advan. Cell Mol. Biol., /: 59-109, 1971. 15. Lyon, M. F. Sex Chromatin and Gene Action in the Mammalian X-Chromosome. Am. J. Ann. Genet., 14: 135-148, 1962. 16. Popescu, N. C., Cioloca, L., Liciu, F., and Encut, I. Chromosomal Analysis of Some Transplanted Tumors Induced by 3-Methylcholanthrene in Golden Hamsters. Intern. J. Cancer, 4: 785-792, 1969. 17. Popescu, N. C., and DiPaolo, J. A. Heterochromatin, Satellite DNA, and Transformed Neoplastic Cells. J. Nati. Cancer Inst., 49: 603-606, 1972. 18. Popescu, N. C., and DiPaolo, J. A. Identification of Syrian Hamster Chromosomes by Acetic-Saline-Giemsa (ASG) and Trypsin Techniques. Cytogenetics, //: 500-507, 1972. 19. Schmid, W. Heterochromatin in Mammals. Arch. Julius Klaus-Stift. Vererbungs forsch. Sozialanthropol. Rassenkyg., 52: 1-59, 1967. 20. Schultz, J. Variegation in Drosophila and the Inert Chromosome Regions. Proc. Nati. Acad. Sei. U. S., 22: 27 33, 1963. 21. Yunis. J. J., and Yasmineh, V. G. Heterochromatin, Satellite DNA and Cell Function. Science, 174: 1200-1210, 1971. Fig. 1. a, karyotype of a cell with 45 chromosomes from Tumor BP45t. Quinacrine mustard stain. In addition to the marker chromosomes (M, and Mj) and the normal fluorescent pattern of this karyotype, one of the B9 chromosomes has a fluorescent, nonbanded, translocated segment (arrow), b, karotype of a cell with 44 chromosomes from Tumor 4NQ016t. Trypsin-Giemsa preparation. The previously described markers include M. which is an A4 chromosome with a nonbanded, darkly stained translocation, c, metaphase of a cell from Tumor BP45t. C band preparation showing consti tutive heterochromatin distribution of normal sex chromosomes and autosomes. In addition, C bands of the marker chromosomes M,, M 2, and M 3 are shown (arrows), d, metaphase of a cell from Tumor 4NQ0161. C band preparation showing that the translocation on the A4 chromosome is heterochromatic. Fig. 2. a, metaphase from 4NQOI61 cell treated with thymidine-3H during early period of S phase. Majority of chromosomes covered with grains including entire X chromosomes and M„. b, identical metaphase after removal of emulsion, c, metaphase of a 4NQOI61 cell exposed to thymidine-3H during a later period of S phase. Short arms of autosomes, I entire X, and one-half of the 2nd one as well as the terminal portion of M. were labeled. d, same metaphase after removal of emulsion. Fig. 3. Autoradiographic G and C band patterns of Markers M3, M6, and M7. Left, comparison of E20 with M3: First row, conventional stain; second row, same chromosomes after autoradiography; third row, G band; and fourth row, C bands. Right, demonstration of the translocation to heterochromatic arm of X to form M,; and the fusion of long arms of Chromosomes Al and A2 to form M, with I centromere. Left to right, G tech nique, DNA replication, and C band technique. 3262 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. Chromosome Constitutive Heterochromatin vif A2 11i lé A3 X ii B6 if l : X M, A2 -4 fi •¿ « i* B7 B6 ;•u Cil 5 u DI6 Hi I7 » M 'r DI' B8 •¿ v 11 it. E20 M B9 f «ft C12 Is s M„A4 * * II I* B7 cu II A3 e« C13 RIO <• .. CM CW E» M3F21 ;- »A K ib 016 " « D19 Ifflr ^fx M,- r —¿ TC v 1C * t; >V i DECEMBER 1973 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. 3263 , " Ma *.* *~ 4 »V T> V xx -w V •¿^ 2C zd M6 lit E20 * I M3 M- 3264 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research. VOL. 33 Distribution of Chromosome Constitutive Heterochromatin of Syrian Hamster Cells Transformed by Chemical Carcinogens J. A. DiPaolo and N. C. Popescu Cancer Res 1973;33:3259-3264. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/12/3259 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.