Original Articles Cytogenet. Cell Genet. 24: 73-83 ( 1979)
The effect of dexamethasone on the karyotype of A9HT cells A. V enetianer. K. B ajnoc/.ky, 1 J. S / idonya, and J. Szahad Institute of Genetics. Biological Research Center. Hungarian Academy of Sciences, Szeged
Abstract. In the majority of glucocorticoid-resistant receptor containing variants of A9HT cells, the number of dot chromosomes was found to be significantly higher than in the parent cells. The higher number of dot chromosomes, however, did not always correlate with the resistance of L cells to dexamethasone. The formation of dot chro mosomes was not the consequence of dexamethasone-induced chromosome breaks. Dotchromosome formation could not be induced by dexamethasone in human diploid cells. It seems likely that dexamethasone can select for A9HT cells with higher numbers of dot chromosomes.
1 Present address: Hospital of Gyor-Sopron County, Laboratory of Genetics, Gyor (Hungary). Request reprints from: Dr. A niko V enetianer, Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences. Szeged. POB 521 (Hungary).
1972; L ippman and T hompson, 1974; Bourgeois and N ewby, 1977). To better understand the mechanism of action of glucocorticoids and steroid resistance, we have isolated and characterized several glucocorticoid-receptor-containing L cell clones (V enetianer et al.. 1978). The responsiveness of the variant clones to dexa methasone (9-a-fluoro-16-a-methyl-l 1^,17«, 21-trihydroxypregna-1,4-diene-/S,21 -dione), a synthetic glucocorticoid, ranged from nearly total to slight resistance. An inter esting feature of these variants is that the majority of the clones show a significant, heritable, and stable increase in the number of so-called dot-like chromosomes. This observation led us to inquire whether (1) the number of dot chromosomes is a conse
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
The rate of growth, uptake of glucose, and incorporation of nucleic acid precursors into macromolecules of transformed mouse fibroblast L cells (E arle. 1943) is inhibited by low doses of glucocorticoids (P ratt and A ronow , 1966). It is possible to obtain sub lines of L cells which are no longer re sponsive to glucocorticoids. The majority of glucocorticoid-resistant fibroblastoid cells are characterized by the absence of or a sharp reduction in glucocorticoid receptors (H ackney et al„ 1970; P ratt and I s h ii ,
74
Venetianer/Bajnoczky/Szidohya/Szabad
Materials and methods Cell culture The L cell lines were derived from fibroblasts transformed in vitro by exposure to methylcholanthrcnc (E arle. 1943). The A9HT cell line was developed by Wiener et al. (1973). From the glucocorticoid-sensitive A9HT cells, several clones with decreased sensitivity to dexamethasone were isolated after chemical mutagenesis. The isolation and characterization of dexamethasone-resistant variant clones have been described elsewhere (Venetianer et al., 1978). A9HT cells were treated with mutagens; clones 11 and 15 were obtained using F.MS, and MNNG was used for 21, 25, and 30. Some of the clones (11, 15, 21. 25, and 30) contained roughly the same number of cytoplasmic receptors per milligram of protein as the sensi tive parent, but they were resistant or less sensi tive to dexamethasone. The glucocorticoid-sensitive LB82 cell line (Littlefield, 1965) and its subline, the dexamethasone-resistant, receptor-containing SI.B82-17 variant (L ippman and T hompson, 1974; Venetianer et al., 1978) were also used for these studies. The growth conditions of the cells are described elsewhere (Venetianer et al., 1978). Chromosome analysis Cultured fibroblast cells in log phase were exposed to Colcemid (0.2 /?
û *> * , * t
3 *
r
» * ^ *>
* 1*90
IrfCH'
I • S medium for 20 days. Dexamethasone inhibits the growth of A9HT cells but does not completely stop cell division in a mass culture. After 10 and 20 days, the number of chromosomes in the dexamethasone-treated cells was counted; although 10 days of treatment did not significantly increase the number of dot chromosomes, 20 days of treatment did. The
average number of dot chromosomes in three experiments (150 metaphases) was 3.46 ± 0.80 (range, 1-5), compared to 2.39 in the untreated A9HT cells. The next question was whether the mo dification in the number of dot chromo somes correlates with the resistance of L cells to dexamethasone. Although clone 15 has decreased sensitivity to dexamethasone, it contains, on the average, even fewer dot chromosomes than A9HT. The dexamethasone-resistant SLB82-17 cells have an average of 0.95 dot chromosomes (range, 0-2) in 100 metaphase (table I), similar to the glucocorticoid-sensitive LB82 parent cell line (Engi i. et al., 1969). These results indicate that although the majority of clones
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
Fig. 3. Karyotype of C-banded chromosomes of clone 30 (chromosomes of five metaphases). Dot chromosomes are placed in the second group.
79
Déxaméthasone and dot-chromosome formation
Table II. Number of males and females from the attached-X test with Drosophila Treatment
F, progeny Males
Females
656 630 580
474 515 451
Z2
Control ( 1 % s u c ro s e ) 10
:i
m
d e x a m e th a s o n e
10 1Mdexamethasone
2.0 0.64
results, it appears that dexamethasone does not effect the frequency of SCE in A9HT cells. The mutagen tests carried out with Dro sophila also gave negative results. In the attached-X test, the frequency of males among the enclosing F| adults did not change (PX1.05) after feeding adult males with 10 :t or 10 1 m dexamethasone (table II). This result clearly shows that dexa methasone does not induce lethal mutations on the sex chromosome of Drosophila, even in very high concentration. In the mosaic test, somatic and germline mosaicism would reveal primarily breakage of and exchange of chromatids and/or point mutations (Mot.i.m and Szabad, 1978). The results of the mosaic test are shown in table III. Control data were obtained from experiments in which larvae were fed standard corn-meal Drosophila food or fed for 4 hours with 1% sucrose. The numbers of w/W" twin. w, and u"'" single spots do not differ from those found in the controls in either experiment A or experiment B. in which larvae were fed for 4 h with 10 ■'* m dexamethasone or were grown on standard corn-meal medium con-
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
with decreased sensitivity to dexamethasone contain significantly higher numbers of dot chromosomes than the sensitive parent cell line, this increase is not a general characte ristic of dexamethasone-resistant L cells. The question was still open whether this effect of dexamethasone was unique for A9HT cells. Because of the prevalent use of dexamethasone in clinical practice, we tested the effect of this synthetic glucocorticoid on the chromosomes of both human fibroblast and human lymphocytes. Fibroblast cultures were prepared from normal human biopsies. After 1 month the cultures were treated with 10 11 and 10 m dexamethasone and checked on the 10th and 20th days of treat ment. Following the dexamethasone treat ment, chromosomes were examined with and without ASG-banding. Neither dexa methasone treatment induced numerical chromosome aberrations in the human fibro blast cells. Similarly, we did not find any chromosome alteration in a phytohemagglu tinin-stimulated short-term culture of pe ripheral blood leukocytes of a patient treated with dexamethasone for 3 months prior to chromosome examination. For investigating the possible mutagenicity of dexamethasone in A9HT cells, the SCE test provides a useful tool (P frry and E vans , 1975). A9HT cells were exposed during two rounds of replication to BrdU and to 10 '' or 10 ’’ m dexamethasone, and the number of SCEs was scored. The frequency of SCEs per chromosome was found to be 0.358 in the absence and 0.4 in the presence of 10 11M dexamethasone (715 and 728 chro mosomes counted, respectively). In another experiment, the frequency of SCEs was found to be 0.63 in the absence and 0.64 in the presence of 10 ’■m dexamethasone (382 and 477 chromosomes counted). From these
Venetianer Bajnoczky/Szidonya Szabad
80
Table 111. Frequency of mosaicism in the
Wing disc
Eye disc
Treatment
Sex Eyes ex- IV / I V ™ w amined twill single spots Control
$ (5
10 dexamethasone for 4 h m
5 X 10-3 m dexamethasone throughout life of larvae
Drosophila test
?
The effect of dexamethasone on the karyotype of A9HT cells A. V enetianer. K. B ajnoc/.ky, 1 J. S / idonya, and J. Szahad Institute of Genetics. Biological Research Center. Hungarian Academy of Sciences, Szeged
Abstract. In the majority of glucocorticoid-resistant receptor containing variants of A9HT cells, the number of dot chromosomes was found to be significantly higher than in the parent cells. The higher number of dot chromosomes, however, did not always correlate with the resistance of L cells to dexamethasone. The formation of dot chro mosomes was not the consequence of dexamethasone-induced chromosome breaks. Dotchromosome formation could not be induced by dexamethasone in human diploid cells. It seems likely that dexamethasone can select for A9HT cells with higher numbers of dot chromosomes.
1 Present address: Hospital of Gyor-Sopron County, Laboratory of Genetics, Gyor (Hungary). Request reprints from: Dr. A niko V enetianer, Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences. Szeged. POB 521 (Hungary).
1972; L ippman and T hompson, 1974; Bourgeois and N ewby, 1977). To better understand the mechanism of action of glucocorticoids and steroid resistance, we have isolated and characterized several glucocorticoid-receptor-containing L cell clones (V enetianer et al.. 1978). The responsiveness of the variant clones to dexa methasone (9-a-fluoro-16-a-methyl-l 1^,17«, 21-trihydroxypregna-1,4-diene-/S,21 -dione), a synthetic glucocorticoid, ranged from nearly total to slight resistance. An inter esting feature of these variants is that the majority of the clones show a significant, heritable, and stable increase in the number of so-called dot-like chromosomes. This observation led us to inquire whether (1) the number of dot chromosomes is a conse
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
The rate of growth, uptake of glucose, and incorporation of nucleic acid precursors into macromolecules of transformed mouse fibroblast L cells (E arle. 1943) is inhibited by low doses of glucocorticoids (P ratt and A ronow , 1966). It is possible to obtain sub lines of L cells which are no longer re sponsive to glucocorticoids. The majority of glucocorticoid-resistant fibroblastoid cells are characterized by the absence of or a sharp reduction in glucocorticoid receptors (H ackney et al„ 1970; P ratt and I s h ii ,
74
Venetianer/Bajnoczky/Szidohya/Szabad
Materials and methods Cell culture The L cell lines were derived from fibroblasts transformed in vitro by exposure to methylcholanthrcnc (E arle. 1943). The A9HT cell line was developed by Wiener et al. (1973). From the glucocorticoid-sensitive A9HT cells, several clones with decreased sensitivity to dexamethasone were isolated after chemical mutagenesis. The isolation and characterization of dexamethasone-resistant variant clones have been described elsewhere (Venetianer et al., 1978). A9HT cells were treated with mutagens; clones 11 and 15 were obtained using F.MS, and MNNG was used for 21, 25, and 30. Some of the clones (11, 15, 21. 25, and 30) contained roughly the same number of cytoplasmic receptors per milligram of protein as the sensi tive parent, but they were resistant or less sensi tive to dexamethasone. The glucocorticoid-sensitive LB82 cell line (Littlefield, 1965) and its subline, the dexamethasone-resistant, receptor-containing SI.B82-17 variant (L ippman and T hompson, 1974; Venetianer et al., 1978) were also used for these studies. The growth conditions of the cells are described elsewhere (Venetianer et al., 1978). Chromosome analysis Cultured fibroblast cells in log phase were exposed to Colcemid (0.2 /?
û *> * , * t
3 *
r
» * ^ *>
* 1*90
IrfCH'
I • S medium for 20 days. Dexamethasone inhibits the growth of A9HT cells but does not completely stop cell division in a mass culture. After 10 and 20 days, the number of chromosomes in the dexamethasone-treated cells was counted; although 10 days of treatment did not significantly increase the number of dot chromosomes, 20 days of treatment did. The
average number of dot chromosomes in three experiments (150 metaphases) was 3.46 ± 0.80 (range, 1-5), compared to 2.39 in the untreated A9HT cells. The next question was whether the mo dification in the number of dot chromo somes correlates with the resistance of L cells to dexamethasone. Although clone 15 has decreased sensitivity to dexamethasone, it contains, on the average, even fewer dot chromosomes than A9HT. The dexamethasone-resistant SLB82-17 cells have an average of 0.95 dot chromosomes (range, 0-2) in 100 metaphase (table I), similar to the glucocorticoid-sensitive LB82 parent cell line (Engi i. et al., 1969). These results indicate that although the majority of clones
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
Fig. 3. Karyotype of C-banded chromosomes of clone 30 (chromosomes of five metaphases). Dot chromosomes are placed in the second group.
79
Déxaméthasone and dot-chromosome formation
Table II. Number of males and females from the attached-X test with Drosophila Treatment
F, progeny Males
Females
656 630 580
474 515 451
Z2
Control ( 1 % s u c ro s e ) 10
:i
m
d e x a m e th a s o n e
10 1Mdexamethasone
2.0 0.64
results, it appears that dexamethasone does not effect the frequency of SCE in A9HT cells. The mutagen tests carried out with Dro sophila also gave negative results. In the attached-X test, the frequency of males among the enclosing F| adults did not change (PX1.05) after feeding adult males with 10 :t or 10 1 m dexamethasone (table II). This result clearly shows that dexa methasone does not induce lethal mutations on the sex chromosome of Drosophila, even in very high concentration. In the mosaic test, somatic and germline mosaicism would reveal primarily breakage of and exchange of chromatids and/or point mutations (Mot.i.m and Szabad, 1978). The results of the mosaic test are shown in table III. Control data were obtained from experiments in which larvae were fed standard corn-meal Drosophila food or fed for 4 hours with 1% sucrose. The numbers of w/W" twin. w, and u"'" single spots do not differ from those found in the controls in either experiment A or experiment B. in which larvae were fed for 4 h with 10 ■'* m dexamethasone or were grown on standard corn-meal medium con-
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 1:31:25 AM
with decreased sensitivity to dexamethasone contain significantly higher numbers of dot chromosomes than the sensitive parent cell line, this increase is not a general characte ristic of dexamethasone-resistant L cells. The question was still open whether this effect of dexamethasone was unique for A9HT cells. Because of the prevalent use of dexamethasone in clinical practice, we tested the effect of this synthetic glucocorticoid on the chromosomes of both human fibroblast and human lymphocytes. Fibroblast cultures were prepared from normal human biopsies. After 1 month the cultures were treated with 10 11 and 10 m dexamethasone and checked on the 10th and 20th days of treat ment. Following the dexamethasone treat ment, chromosomes were examined with and without ASG-banding. Neither dexa methasone treatment induced numerical chromosome aberrations in the human fibro blast cells. Similarly, we did not find any chromosome alteration in a phytohemagglu tinin-stimulated short-term culture of pe ripheral blood leukocytes of a patient treated with dexamethasone for 3 months prior to chromosome examination. For investigating the possible mutagenicity of dexamethasone in A9HT cells, the SCE test provides a useful tool (P frry and E vans , 1975). A9HT cells were exposed during two rounds of replication to BrdU and to 10 '' or 10 ’’ m dexamethasone, and the number of SCEs was scored. The frequency of SCEs per chromosome was found to be 0.358 in the absence and 0.4 in the presence of 10 11M dexamethasone (715 and 728 chro mosomes counted, respectively). In another experiment, the frequency of SCEs was found to be 0.63 in the absence and 0.64 in the presence of 10 ’■m dexamethasone (382 and 477 chromosomes counted). From these
Venetianer Bajnoczky/Szidonya Szabad
80
Table 111. Frequency of mosaicism in the
Wing disc
Eye disc
Treatment
Sex Eyes ex- IV / I V ™ w amined twill single spots Control
$ (5
10 dexamethasone for 4 h m
5 X 10-3 m dexamethasone throughout life of larvae
Drosophila test
?
