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Marker chromosomes of WEHI-3, a murine myelomonocytic cell line
Marker Chromosomes of WEHI-3, A Murine Myelomonocytic Cell Line Sandra Bornstein and Lydia E. McMorrow
ABSTRACT wEHI- is a murine myelomonocytic cell line adapted to growth in vitro. We have examined the chromosome complement of this cell line and found modal chromosome number of 76-79 with four marker chromosomes which were identified by GTG and C banding.
INTRODUCTION Continuously propagated cell lines which manifest growth and regulatory responses characteristic of specialized tissue lineages have been helpful in studying the mechanisms involved in the regulation of neoplastic cells. While several forms of lymphomas (L1210, L5178Y) have existed for many years, a mouse model for myelomonocytic leukemia has been developed relatively recently. The WIG-II-3 line was originally obtained from a myelomonocytic leukemia in BALB/c mice [l]and later adapted to growth in culture [2]. WEHI- cells grow in semisolid agar to form colonies and clusters. Medium in which WEHI- cells have grown contains colony-stimulating factor (CSF) which is capable of stimulating growth of mouse myeloid and monocytic cells from other sources. WEHI- cells respond to growth inhibitory and stimulatory substances and can serve as an in vitro model for studying the regulation of growth of leukemic cells [2]. In this article, we describe the marker chromosomes of the WEHI- cell line because of the potential usefulness of this cell line in the study of leukemia. Cytogenetic characterization may be of value in studies of clonal evolution, tumor transplantation, cell hydridization, or gene localization. MATERIALS
AND METHODS
WEHI- cells were originally obtained from BALB/c mice after induction of myelomonocytic leukemia with mineral oil [l]. The cell line has been carried in culture since 1974 and was a gift from Dr. Peter Ralph, Memorial Sloan-Kettering Institute for Cancer Research, New York. The cells have been propagated in our laboratory by twice weekly subculture (2 x lo5cells/ml) in McCoy’s medium supplemented with
From the Departmentof Pathology, New York University School of Medicine, New York,New York.
Address requests for reprints to: Dr. Sandra Bornstein, Department of Pathology, New York University ofMedicine, 550 First Avenue, New York, NY 10016. Received July 23,1979; accepted September l9,1979. @Elsevier North Holland, Inc., 1980 0165-4608/80/02034505$02.25
Cancer Genetics and Cytogenetics 1, 345-349 (1980)
345
346
Figure 1
S. Bornstein
and L. E. McMorrow
Representative karyotypes of the WEHI- cells (GTG banding).
10% fetal calf serum, penicillin-streptomycin (5000 units-5000 mcg/lOO ml media] and L-glutamine (26.2 mg/lOO ml media). For cytogenetic analysis, the cells were harvested 48 hr after subculture following exposure to colcemid (0.5 mcg/ml) for 3 hr. The cells were swollen with 0.38% potassium choloride and fixed in Carnoy’s fixative (methanol:acetic acid 3 :l). Cells were dropped on slides chilled in methanol and quickly flame-dried over an alcohol lamp. Slides were examined after staining with orcein, G bands by trypsin using Giemsa (GTG), or C bands by barium hydroxide using Giemsa (C banding). Optimal GTG banding was achieved at 24 hr. Slides were placed vertically in a Coplin jar in 0.1% trypsin at room temperature (20 ml trypsin GIBCO (0.25% trypsin) in Hank’s balanced saline solution (HBSS):30 ml phosphate-buffered saline (PBS)) for 90 to 115 set, rinsed quickly in 50% and 100% ethanol, stained in 4% Giemsa stain (2 ml of stock solution) in pH 6.8 phosphate buffer for 90 set, and rinsed quickly in two changes of the same buffer. Slides were blown to complete dryness, dipped in xylene, and cover slipped with Canada Balsam. C banding was obtained off slides after 7 days aging at room temperature, sequentially exposed to 0.2 N HCl for l/2 hr, 0.07% Ba(OH), at 37°C for 7 min, quick rinse in distilled water, 2 X SSC at 65°C for 2 hr, and stained in 4% Giemsa stain (in pH 6.8 buffer) for 5 min. The slides were air dried to complete dryness, placed in xylene for 15 min, and cover slipped in Canada Balsam.
Marker Chromosomes of WEHI- Cell Line
Figure 2
347
Markerchromosomesof WEHI- (GTGbandingon left, C bandingon right).
RESULTS The WEHI- cell line is characterized by a modal number of 76 - 79 (63% of the cells). Most of the loss from the tetraploid state appears to have affected predominantly the larger chromosomes, (#l-4), not all of which can be accounted for by the composition of the markers. There are four large, distinctive markers in virtually every cell (Fig. 1). Two are metacentric and two are larger than normal telocentrics. Banding reveals that these markers are not paired. Therefore, the cell line appears to have undergone marker evolution after becoming tetraploid. The origin of the 4 marker chromosomes has not been unequivocally determined. To the extent to which we have been able, the markers have been characterized as follows (see accompanying diagram and composite, Figs. 2 and 3). Marker 1: Largest Metacentric Two heterochromatic centromeric regions exist. One arm appears to consist of an entire #3 chromosome. The distal region of the other arm appears to be a #16 chromosome (distal to its heterochromatic centromeric region). The origin of the second centromeric region and the proximal half of this arm could not be unequivocally determined.
S. Bornstein
Figure 3
and L. E. McMorrow
Derivation of marker chromosomes of WEHI- cells based on GTG and C banding.
Marker 2: Large Metacentric Two heterochromatic centromeric regions exist. It appears most likely to be an isochromosome of the #lO chromosome. In some of the cells it appears that the pretelomerit dark band on one arm is more distal than that of the other arm. If this difference is significant, it is possible that this arm is, in fact, a #4 chromosome with a deletion distal to the centromeric heterchromatin and proximal to the third dark band. Marker 3: Largest Telocentric A single, centromeric heterochromatic region exists. This chromosome appears to consist of a #lO chromosome with an insertion of the distal 2/3 of chromosome #2 into the distal light region of the #lo.
Marker Chromosomes
of WEHI-
Cell Line
349
Marker 4: Large Telocentric
Like M3, this marker appears to have arisen through an insertion. A break in the distal light region of the #4, and insertion of the distal 2/3 of the #13 could explain its banding pattern. In size this marker was similar to the normal large telocentric chromosomes. Therefore, this chromosome could not be unequivocally identified with C banding and the nature of the centromeric heterochromatic region could not be determined. DISCUSSION
Each of the twenty pairs of chromosomes of the mouse can now be individually identified by means of banding techniques [3]. WEHI- cell line is a murine myelomonocytic leukemia adapted to tissue culture. The original WEHI- tumor had a modal number of 40 with apparently normal chromosomes [2]. After two passages in vivo, two sublines retained this chromosomal pattern, while one subline developed a tetraploid karyotype (mode = 80, all telocentric chromosomes). A fourth subline developed a mode of 78 chromosomes with two metacentric marker chromosomes. Tetraploidy is often associated with malignant transformation both in vivo and in vitro [4 - 61. We have examined the chromosomes of a WEHI- subline propagated in vitro in our laboratory for 1 year, during which time the chromosome complement has remained stable. This subline has a modal chromosome number of 76-79 with four distinctive markers in virtually every cell. Two markers are large metacentric chromosomes, while the other two are large telocentrics. Marker chromosomes have been useful in defining the reproductive history of clones of cells and in identifying and following the evolution of subgroups within a population. In the WEHI- cell line, we have shown that the markers are nonidentical and, therefore, arose after the cell line became tetraploid. Supported by National Institutes of Health Grant CA #22942.
REFERENCES 1. Warner NL, Moore MAS, Metcalf D (1969): A transplantable myelomonocytic leukemia in BALB/c mice: cytology, karyotype and muramidase content. J Nat1 Cancer Inst 43,963 -962. 2. Broxmeyer I-IE, Ralph P (1977): In vitro regulation of a mouse myelomonocytic adapted to culture. Cancer Res 37,3576-3564. 3. Committee on Standarized Genetic Nomenclature mouse, Mus Musculus. J Hered 63,69 - 72.
leukemia line
for Mice (1972): Standard karyotype of the
4. Horibata K, Harris AW (1970): Mouse myelomas and lymphomas 61-77.
in culture. Rxp Cell Res 60,
5. Lehman JM, Defendi V (1970): Changes in deoxyribonucleic acid synthesis Chinese hamster cells infected with simian virus 40. J Virol6,738749.
regulation
in
6. Moriwaki K, Imai I-IT, Yamashita J, Yosida TH (1971): Ploidy fluctuations of mouse plasmacell neoplasm MSPC-1 during serial transplantation. J Nat1 Cancer Inst USA 47,623-637.
ABSTRACT wEHI- is a murine myelomonocytic cell line adapted to growth in vitro. We have examined the chromosome complement of this cell line and found modal chromosome number of 76-79 with four marker chromosomes which were identified by GTG and C banding.
INTRODUCTION Continuously propagated cell lines which manifest growth and regulatory responses characteristic of specialized tissue lineages have been helpful in studying the mechanisms involved in the regulation of neoplastic cells. While several forms of lymphomas (L1210, L5178Y) have existed for many years, a mouse model for myelomonocytic leukemia has been developed relatively recently. The WIG-II-3 line was originally obtained from a myelomonocytic leukemia in BALB/c mice [l]and later adapted to growth in culture [2]. WEHI- cells grow in semisolid agar to form colonies and clusters. Medium in which WEHI- cells have grown contains colony-stimulating factor (CSF) which is capable of stimulating growth of mouse myeloid and monocytic cells from other sources. WEHI- cells respond to growth inhibitory and stimulatory substances and can serve as an in vitro model for studying the regulation of growth of leukemic cells [2]. In this article, we describe the marker chromosomes of the WEHI- cell line because of the potential usefulness of this cell line in the study of leukemia. Cytogenetic characterization may be of value in studies of clonal evolution, tumor transplantation, cell hydridization, or gene localization. MATERIALS
AND METHODS
WEHI- cells were originally obtained from BALB/c mice after induction of myelomonocytic leukemia with mineral oil [l]. The cell line has been carried in culture since 1974 and was a gift from Dr. Peter Ralph, Memorial Sloan-Kettering Institute for Cancer Research, New York. The cells have been propagated in our laboratory by twice weekly subculture (2 x lo5cells/ml) in McCoy’s medium supplemented with
From the Departmentof Pathology, New York University School of Medicine, New York,New York.
Address requests for reprints to: Dr. Sandra Bornstein, Department of Pathology, New York University ofMedicine, 550 First Avenue, New York, NY 10016. Received July 23,1979; accepted September l9,1979. @Elsevier North Holland, Inc., 1980 0165-4608/80/02034505$02.25
Cancer Genetics and Cytogenetics 1, 345-349 (1980)
345
346
Figure 1
S. Bornstein
and L. E. McMorrow
Representative karyotypes of the WEHI- cells (GTG banding).
10% fetal calf serum, penicillin-streptomycin (5000 units-5000 mcg/lOO ml media] and L-glutamine (26.2 mg/lOO ml media). For cytogenetic analysis, the cells were harvested 48 hr after subculture following exposure to colcemid (0.5 mcg/ml) for 3 hr. The cells were swollen with 0.38% potassium choloride and fixed in Carnoy’s fixative (methanol:acetic acid 3 :l). Cells were dropped on slides chilled in methanol and quickly flame-dried over an alcohol lamp. Slides were examined after staining with orcein, G bands by trypsin using Giemsa (GTG), or C bands by barium hydroxide using Giemsa (C banding). Optimal GTG banding was achieved at 24 hr. Slides were placed vertically in a Coplin jar in 0.1% trypsin at room temperature (20 ml trypsin GIBCO (0.25% trypsin) in Hank’s balanced saline solution (HBSS):30 ml phosphate-buffered saline (PBS)) for 90 to 115 set, rinsed quickly in 50% and 100% ethanol, stained in 4% Giemsa stain (2 ml of stock solution) in pH 6.8 phosphate buffer for 90 set, and rinsed quickly in two changes of the same buffer. Slides were blown to complete dryness, dipped in xylene, and cover slipped with Canada Balsam. C banding was obtained off slides after 7 days aging at room temperature, sequentially exposed to 0.2 N HCl for l/2 hr, 0.07% Ba(OH), at 37°C for 7 min, quick rinse in distilled water, 2 X SSC at 65°C for 2 hr, and stained in 4% Giemsa stain (in pH 6.8 buffer) for 5 min. The slides were air dried to complete dryness, placed in xylene for 15 min, and cover slipped in Canada Balsam.
Marker Chromosomes of WEHI- Cell Line
Figure 2
347
Markerchromosomesof WEHI- (GTGbandingon left, C bandingon right).
RESULTS The WEHI- cell line is characterized by a modal number of 76 - 79 (63% of the cells). Most of the loss from the tetraploid state appears to have affected predominantly the larger chromosomes, (#l-4), not all of which can be accounted for by the composition of the markers. There are four large, distinctive markers in virtually every cell (Fig. 1). Two are metacentric and two are larger than normal telocentrics. Banding reveals that these markers are not paired. Therefore, the cell line appears to have undergone marker evolution after becoming tetraploid. The origin of the 4 marker chromosomes has not been unequivocally determined. To the extent to which we have been able, the markers have been characterized as follows (see accompanying diagram and composite, Figs. 2 and 3). Marker 1: Largest Metacentric Two heterochromatic centromeric regions exist. One arm appears to consist of an entire #3 chromosome. The distal region of the other arm appears to be a #16 chromosome (distal to its heterochromatic centromeric region). The origin of the second centromeric region and the proximal half of this arm could not be unequivocally determined.
S. Bornstein
Figure 3
and L. E. McMorrow
Derivation of marker chromosomes of WEHI- cells based on GTG and C banding.
Marker 2: Large Metacentric Two heterochromatic centromeric regions exist. It appears most likely to be an isochromosome of the #lO chromosome. In some of the cells it appears that the pretelomerit dark band on one arm is more distal than that of the other arm. If this difference is significant, it is possible that this arm is, in fact, a #4 chromosome with a deletion distal to the centromeric heterchromatin and proximal to the third dark band. Marker 3: Largest Telocentric A single, centromeric heterochromatic region exists. This chromosome appears to consist of a #lO chromosome with an insertion of the distal 2/3 of chromosome #2 into the distal light region of the #lo.
Marker Chromosomes
of WEHI-
Cell Line
349
Marker 4: Large Telocentric
Like M3, this marker appears to have arisen through an insertion. A break in the distal light region of the #4, and insertion of the distal 2/3 of the #13 could explain its banding pattern. In size this marker was similar to the normal large telocentric chromosomes. Therefore, this chromosome could not be unequivocally identified with C banding and the nature of the centromeric heterochromatic region could not be determined. DISCUSSION
Each of the twenty pairs of chromosomes of the mouse can now be individually identified by means of banding techniques [3]. WEHI- cell line is a murine myelomonocytic leukemia adapted to tissue culture. The original WEHI- tumor had a modal number of 40 with apparently normal chromosomes [2]. After two passages in vivo, two sublines retained this chromosomal pattern, while one subline developed a tetraploid karyotype (mode = 80, all telocentric chromosomes). A fourth subline developed a mode of 78 chromosomes with two metacentric marker chromosomes. Tetraploidy is often associated with malignant transformation both in vivo and in vitro [4 - 61. We have examined the chromosomes of a WEHI- subline propagated in vitro in our laboratory for 1 year, during which time the chromosome complement has remained stable. This subline has a modal chromosome number of 76-79 with four distinctive markers in virtually every cell. Two markers are large metacentric chromosomes, while the other two are large telocentrics. Marker chromosomes have been useful in defining the reproductive history of clones of cells and in identifying and following the evolution of subgroups within a population. In the WEHI- cell line, we have shown that the markers are nonidentical and, therefore, arose after the cell line became tetraploid. Supported by National Institutes of Health Grant CA #22942.
REFERENCES 1. Warner NL, Moore MAS, Metcalf D (1969): A transplantable myelomonocytic leukemia in BALB/c mice: cytology, karyotype and muramidase content. J Nat1 Cancer Inst 43,963 -962. 2. Broxmeyer I-IE, Ralph P (1977): In vitro regulation of a mouse myelomonocytic adapted to culture. Cancer Res 37,3576-3564. 3. Committee on Standarized Genetic Nomenclature mouse, Mus Musculus. J Hered 63,69 - 72.
leukemia line
for Mice (1972): Standard karyotype of the
4. Horibata K, Harris AW (1970): Mouse myelomas and lymphomas 61-77.
in culture. Rxp Cell Res 60,
5. Lehman JM, Defendi V (1970): Changes in deoxyribonucleic acid synthesis Chinese hamster cells infected with simian virus 40. J Virol6,738749.
regulation
in
6. Moriwaki K, Imai I-IT, Yamashita J, Yosida TH (1971): Ploidy fluctuations of mouse plasmacell neoplasm MSPC-1 during serial transplantation. J Nat1 Cancer Inst USA 47,623-637.