Appearance and Evolution of the Specific Chromosomal Rearrangements Associated with Malignant Transformation of Mouse m5S Cells Seiji Kodama, Yutaka Okumura, Kenshi Komatsu, and Masao S. Sasaki
ABSTRACT: Chromosomal alterations were studied during the acquisition of malignant phenotypes in two karyotypically distinct ceils isolated from transformed loci induced by x-irradiation in mouse m5S cells. Because the transformants, despite loci origin, showed low ability to grow in agar, they were cultured in vitro with serial transfer schedules to allow further cell generations and assayed for anchorage independence (AI) at each passage level, The Al frequency increased with the cell doubling numbers. Chromosome analysis showed that a focus was one cell origin, but the transformants showed karyotypic instability during cell proliferation, giving rise to the rearrangements clustered in the distal region of the specific chromosomes. These rearrangements appeared to be directed toward the acquisition of malignant phenotypes. Analysis of the types and sites of rearrangements indicated that a mechanism exists that induces frequent rearrangements of the specific region of a chromosome during the process of transformation into the malignant state. INTRODUCTION W i t h the advances in cancer cytogenetics coupled with techniques in molecular biology, a c c u m u l a t e d evidence indicates that chromosomal m e c h a n i s m s play a significant role in the d e v e l o p m e n t of cancer [1, 2], including oncogene activation by a site-specific translocation [3-6), oncogene amplification by formation of homogen e o u s l y staining regions (HSRs) or (double minutes) (dmin) [7, 8], and allele loss of tumor suppressor gene by chromosomal segregation or mitotic recombination [9]. These c h r o m o s o m a l changes are believed to have a crucial effect on the malignant p h e n o t y p e of cancer cells. Therefore, the m e c h a n i s m of these genome rearrangements is particularly intriguing in cancer research. One approach to studying such a mechanism is d e v e l o p m e n t of suitable experimental system in w h i c h both changes in malignant p h e n o t y p e s and in chromosomes are analyzed. We established an assay system for in vitro transformation using the n e a r - d i p l o i d mouse cell line m5S, w h i c h is highly sensitive to postconfluence inhibition of cell division, and showed the involvement of a n o n r a n d o m chromosome change in xFrom the Department of Radiation Biophysics, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki (S. K., O., Y., K. K.), and Radiation BiologyCenter, Kyoto University, Kyoto (M S. S.), Japan. Address reprint requests to: Seifi Kodama, Department of Radiation Biophysics, Atomic Disease Institutue, Nagasaki University School of Medicine, Sakamoto-machi, Nagasaki 852, Japan. Received January 12, 1990; accepted April 25, 1990
185 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 53:185-197 (1991) 0165-4608/91/$03.50
186
'~. Kod~H.. et ~1
ray-induced cell transformation [10, 11]. This chromosome change,, the dimimltion of chromosome 13 (C2-ter), was critical in forming transformed foci, but was not sufficient to acquire further malignant phenotypes, such as anchorage independence (AI) and tumorigenicity. The acquisition of the latter two phenotypes was associated with a secondary chromosomal change--the addition of an extra chromosomal segment apparent as an abnormally banded region. To investigate further the characteristics of transformants with diminution of chromosome 13, we examined malignant phenotypes and their chromosome stability during cell cultures with successive transfer. We showed that they acquired malignant phenotypes with increasing population doubling numbers and that the chromosome rearrangements occurred at the specific sites of the specific chromosmnes.
MATERIALS AND METHODS Cell Culture Two focus-derived transformants with diminution of chromosome 13 induced with x-rays from parental m5S/1M cells, clone 6310 and clone 4103, were chosen for this study to evaluate the involvement of chromosomal alterations during malignant progression. Parental m5S/1M cells were used as control. All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2. The culture medium was a-modified minimum essential medium supplemented with 10% heat-inactivated fetal calf serum, 20 mM HEPES, penicillin (100 U/ml), and streptomycin (100 /xg/ml). Two types of subculture schedules were applied as follows: 1) cells were transferred by inoculating 105 cells/60-mm dish every 3 days, which allowed cells to remain in an exponentially growing phase; and 2) cells were maintained in an exponentially growing phase by the same subculture schedule described above except for the last passage, in which cells were inoculated at a density of 4 x 105 cells per dish and then held for 15 days in plateau phase; with medium change performed every 3 days at the last passage. In this subculture, cells were expected to experience both an exponentially growing phase and a plateau phase.
Anchorage Independence Assay To evaluate malignant phenotypes of transformants, the AI was assayed at each passage in culture. This assay method has been described previously [11]. Aliquots (5-ml) of 2% agar medium were distributed into 60-mm plastic dishes as the bottom layer. Cell suspensions (2.5 × 104 cells/ml) at 37°C were prepared with liquid medium; 2 vol was then mixed with 1 vol 1% agar medium prewarmed to 40°C. A 3-ml aliquot of this mixture (0.33%) was gently overlaid onto the bottom layer in each dish. Twenty-four hours later, 1 ml liquid medium was added to the culture. The number of colonies was counted under a dissection miscroscope after 3-week incubation; 5 x 105 cells were assayed for each cell line.
Chromosome Analysis The procedures of chromosome preparations were essentially the same as those described previously [11]. Mitotic cells were harvested by incubation for 2 hours with Colcemid (30 ng/ml). The cells were treated with 75 mM KC1 for 40 minutes and fixed in methanol : acetic acid (3 : 1). Air-dried chromosome slides were baked at 100°C for 20 minutes, treated with 0.025% trypsin for 10-15 seconds, and stained with 2%
18 7
Chromosomal Rearrangements and Transformation
10-2 j w ~
O
v/w~
/ ~ 7
C
v/
t" ¢~.
~ v
/
1o-3 Q; 01
.
v
ac-
u c
ABSTRACT: Chromosomal alterations were studied during the acquisition of malignant phenotypes in two karyotypically distinct ceils isolated from transformed loci induced by x-irradiation in mouse m5S cells. Because the transformants, despite loci origin, showed low ability to grow in agar, they were cultured in vitro with serial transfer schedules to allow further cell generations and assayed for anchorage independence (AI) at each passage level, The Al frequency increased with the cell doubling numbers. Chromosome analysis showed that a focus was one cell origin, but the transformants showed karyotypic instability during cell proliferation, giving rise to the rearrangements clustered in the distal region of the specific chromosomes. These rearrangements appeared to be directed toward the acquisition of malignant phenotypes. Analysis of the types and sites of rearrangements indicated that a mechanism exists that induces frequent rearrangements of the specific region of a chromosome during the process of transformation into the malignant state. INTRODUCTION W i t h the advances in cancer cytogenetics coupled with techniques in molecular biology, a c c u m u l a t e d evidence indicates that chromosomal m e c h a n i s m s play a significant role in the d e v e l o p m e n t of cancer [1, 2], including oncogene activation by a site-specific translocation [3-6), oncogene amplification by formation of homogen e o u s l y staining regions (HSRs) or (double minutes) (dmin) [7, 8], and allele loss of tumor suppressor gene by chromosomal segregation or mitotic recombination [9]. These c h r o m o s o m a l changes are believed to have a crucial effect on the malignant p h e n o t y p e of cancer cells. Therefore, the m e c h a n i s m of these genome rearrangements is particularly intriguing in cancer research. One approach to studying such a mechanism is d e v e l o p m e n t of suitable experimental system in w h i c h both changes in malignant p h e n o t y p e s and in chromosomes are analyzed. We established an assay system for in vitro transformation using the n e a r - d i p l o i d mouse cell line m5S, w h i c h is highly sensitive to postconfluence inhibition of cell division, and showed the involvement of a n o n r a n d o m chromosome change in xFrom the Department of Radiation Biophysics, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki (S. K., O., Y., K. K.), and Radiation BiologyCenter, Kyoto University, Kyoto (M S. S.), Japan. Address reprint requests to: Seifi Kodama, Department of Radiation Biophysics, Atomic Disease Institutue, Nagasaki University School of Medicine, Sakamoto-machi, Nagasaki 852, Japan. Received January 12, 1990; accepted April 25, 1990
185 © 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 53:185-197 (1991) 0165-4608/91/$03.50
186
'~. Kod~H.. et ~1
ray-induced cell transformation [10, 11]. This chromosome change,, the dimimltion of chromosome 13 (C2-ter), was critical in forming transformed foci, but was not sufficient to acquire further malignant phenotypes, such as anchorage independence (AI) and tumorigenicity. The acquisition of the latter two phenotypes was associated with a secondary chromosomal change--the addition of an extra chromosomal segment apparent as an abnormally banded region. To investigate further the characteristics of transformants with diminution of chromosome 13, we examined malignant phenotypes and their chromosome stability during cell cultures with successive transfer. We showed that they acquired malignant phenotypes with increasing population doubling numbers and that the chromosome rearrangements occurred at the specific sites of the specific chromosmnes.
MATERIALS AND METHODS Cell Culture Two focus-derived transformants with diminution of chromosome 13 induced with x-rays from parental m5S/1M cells, clone 6310 and clone 4103, were chosen for this study to evaluate the involvement of chromosomal alterations during malignant progression. Parental m5S/1M cells were used as control. All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2. The culture medium was a-modified minimum essential medium supplemented with 10% heat-inactivated fetal calf serum, 20 mM HEPES, penicillin (100 U/ml), and streptomycin (100 /xg/ml). Two types of subculture schedules were applied as follows: 1) cells were transferred by inoculating 105 cells/60-mm dish every 3 days, which allowed cells to remain in an exponentially growing phase; and 2) cells were maintained in an exponentially growing phase by the same subculture schedule described above except for the last passage, in which cells were inoculated at a density of 4 x 105 cells per dish and then held for 15 days in plateau phase; with medium change performed every 3 days at the last passage. In this subculture, cells were expected to experience both an exponentially growing phase and a plateau phase.
Anchorage Independence Assay To evaluate malignant phenotypes of transformants, the AI was assayed at each passage in culture. This assay method has been described previously [11]. Aliquots (5-ml) of 2% agar medium were distributed into 60-mm plastic dishes as the bottom layer. Cell suspensions (2.5 × 104 cells/ml) at 37°C were prepared with liquid medium; 2 vol was then mixed with 1 vol 1% agar medium prewarmed to 40°C. A 3-ml aliquot of this mixture (0.33%) was gently overlaid onto the bottom layer in each dish. Twenty-four hours later, 1 ml liquid medium was added to the culture. The number of colonies was counted under a dissection miscroscope after 3-week incubation; 5 x 105 cells were assayed for each cell line.
Chromosome Analysis The procedures of chromosome preparations were essentially the same as those described previously [11]. Mitotic cells were harvested by incubation for 2 hours with Colcemid (30 ng/ml). The cells were treated with 75 mM KC1 for 40 minutes and fixed in methanol : acetic acid (3 : 1). Air-dried chromosome slides were baked at 100°C for 20 minutes, treated with 0.025% trypsin for 10-15 seconds, and stained with 2%
18 7
Chromosomal Rearrangements and Transformation
10-2 j w ~
O
v/w~
/ ~ 7
C
v/
t" ¢~.
~ v
/
1o-3 Q; 01
.
v
ac-
u c
