SHORT COMMUNICATIONS Apparent Lack of Telomere Sequences on Double Minute Chromosomes C. C. Lin, Julianne Meyne, R. Sasi, and R. K. Moyzis
Double minute chromosomes (dmins) were first seen in the metaphase spreads of a patient with carcinoma of the lung [1]. The minute pairs of apparently acentric extrachromosomal chromatin bodies were later found in a number of established tumor cell lines, as well as in other primary tumors [2, 3]. It is now known that dmins and their counterparts, homogeneously staining regions (HSR) are the cytogenetic manifestation of gene amplification [4-6]; dmins replicate autonomously once per cell cycle [7]. By PCC technique most dmins observed in late G1 cells appeared to be single. Thus, dmin can invariably assume a double configuration after the S phase [8]. Electron microscopy showed that dmins lack normal kinetochore structures [9]. This was confirmed by Haaf and Schmid [10] using antikinetochore antibody analysis. Thus, dmins are believed to segregate randomly into daughter cells, and their number may vary from several to hundreds per cell (Fig. 1). Several lines of evidence indicate that dmins have a circular structure. Ultrastructural studies have found no free ends in dmin preparations [11]. Recently, untreated dmins were shown not to enter a pulsed field gel until treated with either ~,-irradiation, DNase I, or restriction enzymes [12]. This is indicative of a circular molecule being linearized. We report that dmins also apparently lack telomeric sequences. Routine air-drying technique of chromosome preparation was performed on cells from a human colon carcinoma cell line (Cole 320-DM) [13]. Metaphase spreads were hybridized in situ to the biotinylated telomeric sequences (TTAGGG)n as a probe [14]. The in situ hybridization was conducted in 2 × SSC/30% (vol/vol) formamide at 37°C as described by Moyzis et al [15]. After a single amplification of the hybridization signal with antiavidin antibody, the chromosomes were counter-stained with propidium iodide (1 ~g/ml) in antifade solution [16]. Photographs were taken with Kodacolor 400 film using a Zeiss Axioplan epifluorescent microscope. Bright fluorescein isothiocyanate (FITC) fluorescent signals were observed in the telomeric regions of regular chromosomes (bright yellow dots in the terminal end of chromosome arms). However, no yellow FITC fluorescent dots
From the Department of Pathology and LaboratoryMedicine, University of Alberta and University of Alberta Hospital, Edmonton, Alberta, Canada (C. C. L., R. S.), and Genetics Group, Los Alamos National Laboratory, Los Alamos New Mexico (J. M., R. K. M.) Address reprints requests to: C. C. Lin, Ph.D., Cytogenetics Laboratory, 5B4.49 W.C. Mackenzie Health Science Centre, University of Alberta Hospitals, 8440 112 St., Edmonton, Alberta, Canada, T6G 2B7. Received October 3, 1989; accepted October 19, 1989.
271 © 1990 Elsevier Science Publishing Co., Inc. 655 A v e n u e of the Americas, New York, NY 10010
Cancer Genet Cytogenet 4 8 : 2 7 1 - 2 7 4 (1990) 0165-4608/90/$03.50
272
c . c . L i n e t el.
Figure 1 A metaphase spread of Colo 300-DM cell showing several hundred double minute chromosomes.
were observed on the dmin, which appeared orange-red in color {Fig. 2}. This finding indicates that the d m i n s lack detectable amounts of teloIneric sequences, suggesting that they do not have the telomeric structure as observed in normal chromosomes. This result has been confirmed in cell lines derived from breast tumors and an additional colon carcinoma (data not shown). In situ hybridization has been used successfully to detect telomere sequences in very tiny accessory chromosomes in h u m a n cells [17] and in microchromosomes of birds and reptiles [18]. Some of these chromosomes are about the same size as large drain. Thus, the size of the d m i n should not affect the outcome of the hybridization. The apparent lack of telomere sequence in d m i n must be confirmed by molecular methods, e.g., hybridization of the telomere sequence to isolated d m i n DNA, before it can be determined that d m i n s do not contain any telomere sequences. This observation does, however, lend further support to the notion that" a drain is circular in structure, a m i n u t e acentric ring chromosome.
The authors thank the Medical Research Council of Canada (Grant No. MA8588 to C. C. L.} and the Department of Energy (Contract No. 4718/F18 to R. K. M.), and from Los Alamos National Laboratory for support (to J. M.) for this study.
Lack of T e l o m e r e S e q u e n c e s on D o u b l e M i n u t e C h r o m o s o m e s
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F i g u r e 2 A metaphase spread of Colo 300-DM cell hybridized in situ to biotin-labeled (GGGTTA)7.(TAACCC)7 oligomers. Chromosomes were counterstained with propidium iodide after reaction with fluorescein-labeled avidin and one amplification with avidin antibody to detect the biotinylated DNA sequences. Bright yellow fluorescent dots appeared in the terminal regions of chromosome arms of regular chromosomes. No fluorescein isothiocyanate fluorescence signal of yellow dots could be detected in the double minute chromosome (some indicated by
arrows).
REFERENCES 1. Spriggs AI, Boddington MM, Clarke CM (1982): Chromosomes of human cancer cells. Br Med J 2:1431-1435. 2. Balaban-Malenbaum G, Gilbert F (1977): Double minute chromosomes and homogeneously staining regions in chromosomes of a human neuroblastoma cell line. Science 198:739-741. 3. Barker PE (1982): Double minutes in human tumor cells. Cancer Genet Cytogenet 5:81-94. 4. Kaufman RJ, Brown PC, Schimke RT (1979): Amplified dihydrofolate reductase genes in unstably methotrexate resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci USA 76:5669-5673. 5. Lin CC, Alitalo K, Schwab M, George D, Varmus HE, Bishop JM (1985): Evolution of karyotypic abnormalities and c-myc oncogene amplification in human colonic carcinoma cell line. Chromosom~i 92:11-15. 6. Misawa S, Staal SP, Testa JR (1987): Amplification of the c-myc oncogene is associated with an abnormally banded region on chromosome 8 or double minute chromosomes in two HL-60 human leukemic sublines. Cancer Genet Cytogenet 28:127-136. 7. Barker PE, Drwinga HL, Hittelman WN, Maddox A-M (1980): Double minutes replicate once during S phase of the cell cycle. Exp Cell Res 130:353-360. 8. Takayama S, Uwaike Y (1988): Analysis of the replication mode of double minutes using the PCC technique combined with BrdUrd .labeling. Chromosoma 97:198-203. 9. Rattner JB, Lin CC (1984): Ultrastructural organization of double minute chromosomes and HSR regions in human colon carcinoma cells. Cytogenet Cell Genet 38:176-181.
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c.c.
Lin et al.
10. Haaf T, Schmid M (1988): Analysis of double minutes and double minute-like chromatin in human and murine tumor cells using antikinetochore antibodies. Cancer Genet Cytogenet 30:73-82. 11. Hamkalo BA, Farnham PJ, Johston R, Schimke RT (1985): Ultrastructural features of minute chromosomes in methotrexate-resistant mouse 3T3 cell line. Proc Natl Acad Sci USA 82:1126-1130. 12. van der Btiek AM, Lincke CR, Boist P (1988): Circular DNA of 3T6R50 double minute chromosomes. Nucleic Acids Res 16:4841-4851. 13. Quinn LA, Moore GE, Morgan PT, Woods LK (1979): Cell lines from human colon carcinoma with unusual cell products, double minutes, and homogeneously staining regions. Cancer Res 39:4914-4924. 14. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu J-R (1988): A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomers of human chromosomes. Proc Natl Acad Sci USA 85:6622-6626. 15. Moyzis RK, Albright KL, Bartholdi MF, Cram LS, Deaven LL, Hildebrand CE, Joste NE, Longmire JL, Meyne J, Robinson TS (1987): Human chromosome-specific repetitive DNA sequences: Novel markers for genetic analysis. Chromosoma 95:375-386. 16. Johnson GD, Aranjo GM (1981): A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Meth 43:349-350. 17. Lin CC, Meyne J, Sasi R, Bowen P, Unger T, Tainaka T, Hadra A and Hoo JJ (1990}: Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,X/46,X, + MAR by use of chromosome specific DNA probes. Am J Med Genet {in press). 18. Meyne J, Ratliff RL, Moyzis RK (1989): Conservation of the human telomere sequence (TTAGGG)n among vertibrates. Proc Nat| Acad Sci USA 86:7049-7053.
Double minute chromosomes (dmins) were first seen in the metaphase spreads of a patient with carcinoma of the lung [1]. The minute pairs of apparently acentric extrachromosomal chromatin bodies were later found in a number of established tumor cell lines, as well as in other primary tumors [2, 3]. It is now known that dmins and their counterparts, homogeneously staining regions (HSR) are the cytogenetic manifestation of gene amplification [4-6]; dmins replicate autonomously once per cell cycle [7]. By PCC technique most dmins observed in late G1 cells appeared to be single. Thus, dmin can invariably assume a double configuration after the S phase [8]. Electron microscopy showed that dmins lack normal kinetochore structures [9]. This was confirmed by Haaf and Schmid [10] using antikinetochore antibody analysis. Thus, dmins are believed to segregate randomly into daughter cells, and their number may vary from several to hundreds per cell (Fig. 1). Several lines of evidence indicate that dmins have a circular structure. Ultrastructural studies have found no free ends in dmin preparations [11]. Recently, untreated dmins were shown not to enter a pulsed field gel until treated with either ~,-irradiation, DNase I, or restriction enzymes [12]. This is indicative of a circular molecule being linearized. We report that dmins also apparently lack telomeric sequences. Routine air-drying technique of chromosome preparation was performed on cells from a human colon carcinoma cell line (Cole 320-DM) [13]. Metaphase spreads were hybridized in situ to the biotinylated telomeric sequences (TTAGGG)n as a probe [14]. The in situ hybridization was conducted in 2 × SSC/30% (vol/vol) formamide at 37°C as described by Moyzis et al [15]. After a single amplification of the hybridization signal with antiavidin antibody, the chromosomes were counter-stained with propidium iodide (1 ~g/ml) in antifade solution [16]. Photographs were taken with Kodacolor 400 film using a Zeiss Axioplan epifluorescent microscope. Bright fluorescein isothiocyanate (FITC) fluorescent signals were observed in the telomeric regions of regular chromosomes (bright yellow dots in the terminal end of chromosome arms). However, no yellow FITC fluorescent dots
From the Department of Pathology and LaboratoryMedicine, University of Alberta and University of Alberta Hospital, Edmonton, Alberta, Canada (C. C. L., R. S.), and Genetics Group, Los Alamos National Laboratory, Los Alamos New Mexico (J. M., R. K. M.) Address reprints requests to: C. C. Lin, Ph.D., Cytogenetics Laboratory, 5B4.49 W.C. Mackenzie Health Science Centre, University of Alberta Hospitals, 8440 112 St., Edmonton, Alberta, Canada, T6G 2B7. Received October 3, 1989; accepted October 19, 1989.
271 © 1990 Elsevier Science Publishing Co., Inc. 655 A v e n u e of the Americas, New York, NY 10010
Cancer Genet Cytogenet 4 8 : 2 7 1 - 2 7 4 (1990) 0165-4608/90/$03.50
272
c . c . L i n e t el.
Figure 1 A metaphase spread of Colo 300-DM cell showing several hundred double minute chromosomes.
were observed on the dmin, which appeared orange-red in color {Fig. 2}. This finding indicates that the d m i n s lack detectable amounts of teloIneric sequences, suggesting that they do not have the telomeric structure as observed in normal chromosomes. This result has been confirmed in cell lines derived from breast tumors and an additional colon carcinoma (data not shown). In situ hybridization has been used successfully to detect telomere sequences in very tiny accessory chromosomes in h u m a n cells [17] and in microchromosomes of birds and reptiles [18]. Some of these chromosomes are about the same size as large drain. Thus, the size of the d m i n should not affect the outcome of the hybridization. The apparent lack of telomere sequence in d m i n must be confirmed by molecular methods, e.g., hybridization of the telomere sequence to isolated d m i n DNA, before it can be determined that d m i n s do not contain any telomere sequences. This observation does, however, lend further support to the notion that" a drain is circular in structure, a m i n u t e acentric ring chromosome.
The authors thank the Medical Research Council of Canada (Grant No. MA8588 to C. C. L.} and the Department of Energy (Contract No. 4718/F18 to R. K. M.), and from Los Alamos National Laboratory for support (to J. M.) for this study.
Lack of T e l o m e r e S e q u e n c e s on D o u b l e M i n u t e C h r o m o s o m e s
273
F i g u r e 2 A metaphase spread of Colo 300-DM cell hybridized in situ to biotin-labeled (GGGTTA)7.(TAACCC)7 oligomers. Chromosomes were counterstained with propidium iodide after reaction with fluorescein-labeled avidin and one amplification with avidin antibody to detect the biotinylated DNA sequences. Bright yellow fluorescent dots appeared in the terminal regions of chromosome arms of regular chromosomes. No fluorescein isothiocyanate fluorescence signal of yellow dots could be detected in the double minute chromosome (some indicated by
arrows).
REFERENCES 1. Spriggs AI, Boddington MM, Clarke CM (1982): Chromosomes of human cancer cells. Br Med J 2:1431-1435. 2. Balaban-Malenbaum G, Gilbert F (1977): Double minute chromosomes and homogeneously staining regions in chromosomes of a human neuroblastoma cell line. Science 198:739-741. 3. Barker PE (1982): Double minutes in human tumor cells. Cancer Genet Cytogenet 5:81-94. 4. Kaufman RJ, Brown PC, Schimke RT (1979): Amplified dihydrofolate reductase genes in unstably methotrexate resistant cells are associated with double minute chromosomes. Proc Natl Acad Sci USA 76:5669-5673. 5. Lin CC, Alitalo K, Schwab M, George D, Varmus HE, Bishop JM (1985): Evolution of karyotypic abnormalities and c-myc oncogene amplification in human colonic carcinoma cell line. Chromosom~i 92:11-15. 6. Misawa S, Staal SP, Testa JR (1987): Amplification of the c-myc oncogene is associated with an abnormally banded region on chromosome 8 or double minute chromosomes in two HL-60 human leukemic sublines. Cancer Genet Cytogenet 28:127-136. 7. Barker PE, Drwinga HL, Hittelman WN, Maddox A-M (1980): Double minutes replicate once during S phase of the cell cycle. Exp Cell Res 130:353-360. 8. Takayama S, Uwaike Y (1988): Analysis of the replication mode of double minutes using the PCC technique combined with BrdUrd .labeling. Chromosoma 97:198-203. 9. Rattner JB, Lin CC (1984): Ultrastructural organization of double minute chromosomes and HSR regions in human colon carcinoma cells. Cytogenet Cell Genet 38:176-181.
274
c.c.
Lin et al.
10. Haaf T, Schmid M (1988): Analysis of double minutes and double minute-like chromatin in human and murine tumor cells using antikinetochore antibodies. Cancer Genet Cytogenet 30:73-82. 11. Hamkalo BA, Farnham PJ, Johston R, Schimke RT (1985): Ultrastructural features of minute chromosomes in methotrexate-resistant mouse 3T3 cell line. Proc Natl Acad Sci USA 82:1126-1130. 12. van der Btiek AM, Lincke CR, Boist P (1988): Circular DNA of 3T6R50 double minute chromosomes. Nucleic Acids Res 16:4841-4851. 13. Quinn LA, Moore GE, Morgan PT, Woods LK (1979): Cell lines from human colon carcinoma with unusual cell products, double minutes, and homogeneously staining regions. Cancer Res 39:4914-4924. 14. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu J-R (1988): A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomers of human chromosomes. Proc Natl Acad Sci USA 85:6622-6626. 15. Moyzis RK, Albright KL, Bartholdi MF, Cram LS, Deaven LL, Hildebrand CE, Joste NE, Longmire JL, Meyne J, Robinson TS (1987): Human chromosome-specific repetitive DNA sequences: Novel markers for genetic analysis. Chromosoma 95:375-386. 16. Johnson GD, Aranjo GM (1981): A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Meth 43:349-350. 17. Lin CC, Meyne J, Sasi R, Bowen P, Unger T, Tainaka T, Hadra A and Hoo JJ (1990}: Determining the origins and the structural aberrations of small marker chromosomes in two cases of 45,X/46,X, + MAR by use of chromosome specific DNA probes. Am J Med Genet {in press). 18. Meyne J, Ratliff RL, Moyzis RK (1989): Conservation of the human telomere sequence (TTAGGG)n among vertibrates. Proc Nat| Acad Sci USA 86:7049-7053.
