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Spontaneous chromosome fragility in chorionic villus cells
Early Human Development,
26 (1991) 93-99
93
Elsevier Scientific Publishers Ireland Ltd. EHD 01169
Spontaneous
chromosome fragility villus cells
in chorionic
L. Miguez, C. Fuster, M.M. PCrez, R. Mir6 and J. Egozcue Departamenr
de Biologia Cellular i Fisiologia, Universitat Autbnoma de Barcelona, Barcelona (Spain)
E-08193. Bellaterra.
(Received 18 March 1991; accepted 7 June 1991)
Summary Human fragile sites are only very rarely expressed spontaneously. In this paper we report the presence of non-random spontaneous chromosome lesions (CL) in chorionic villus samples and their coincidence with fragile site (FS) bands. The average number ofCL was about 9% both in RPMI-1640 and in Chang media. To determine any possible influence of external factors other than culture media, the results were grouped according to age of gestation. No differences were observed among the different groups. A total of 101 chromosome lesions could be precisely identified by sequential Leishman Staining/Wright G-banding; 76.2% of them coincided with FS-bands. The most affected region was at lq12-lq21.1 (15.8% of total CL); other FS with a clustering of breakpoints in our study were 1~36, lq44, 2q37, 3~24, 3q27, lOq22 and 16q23. These results suggest that spontaneous expression of some FS could be a characteristic of embryonic tissues.
chromosome fragility; chromosome
lesions; embryonic tissue; chorionic villus cells
Introduction
Fragile sites on human chromosomes are weak points in the genome where nonrandom breaks or gaps can be induced by certain culture conditions. It is known that spontaneous gaps and breaks detected in control lymphocytes are concentrated at high-expression common fragile sites [ 1,2]. Correspondence to: Luz Miguez, Departament de Biologia Cellular i Fisiologia, Subunitat de Biologia Cellular, Facultat de Ciencies, Universitat Autbnoma de Barcelona, E-08 193 Bellaterra Barcelona (Spain)
0378-3782/91/$3.50 0 1991 Elsevier Scientitic Publishers Ireland Ltd. Published and Printed in Ireland
94
Spontaneous expression of fragile sites has been found in first-trimester placental tissues and molar tissues [3] in human sperm chromosomes [4], in cultured bone marrow cells from two patients with hematologic disorders [5] and in a case of epidermoid carcinoma of the esophagus [6]. In this paper we report the presence of non-random, spontaneous chromosome lesions in chorionic villus samples and their coincidence with FS accepted by the committee on cytogenetic markers [7]. Material and Methods
Chorionic villus samples were obtained from 244 cases, with gestational ages of 8-14 weeks (200) 15-19 weeks (32) or more than 20 weeks (8); in four cases the gestational age was not exactly known. Tissues samples were cultured for 24 h using a modification of the method of Simoni et al. [8]; colcemid (final concentration 0.1 &ml) was added to cultures 50 min before harvesting, and preparations made on prewarmed slides (40°C) after samples had been kept overnight at 4°C. CVS were grown in RPMI-1640 (175 cases) or Chang media (69 cases). A sequential technique Leishman-Wright G-Banding of chromosomes from chorionic villus samples to characterize chromosome lesions was used [9]. The preparations were stained with Leishman (1:4 in Leishman buffer) for 30 min, washed with distilled water and photographed. Later on, preparations, aged for 3-4 days at room temperature, were destained for 1 min each in methanol and distilled water. Immediately afterwards, preparations were incubated for 3-5 min in 2 x SSC at 65°C washed with distilled water, air dried and stained for 3 min with Wright’s (1:3 in Sorensen’s buffer, pH 6.8). Results
The frequencies of spontaneous chromosome gaps and breaks detected in our study are indicated in Table I. The average number of chromosome lesions (CL) was 9.0% in RPMI-1640 medium and 9.3% in Chang medium. Of the 199 gaps and breaks observed in the cells cultured in RPMI-1640 medium, 23.6% (471199 CL) were of the chromosome type and 76.4% (1521199) were of the chromatid type: the corresponding figures for the cells cultured in Chang medium were 21.8% (17/78 CL) and 78.2% (61/78 CL). The use of the sequential technique, Leishman staining Gbanding, allowed the precise localization of chromosome bands affected by chromosome lesions (Fig. 1). Due to the quality of CV chromosomes, only 36”/0of preparations could be successfully banded to allow the exact localization of chromosome lesions; the total number of lesions exactly identified was 101. The distribution of the CL identified was non-random (Fig. 2): 76.2% of them coincided with fragile sites (77/101 CL). Lesions at bands lq12 or q21.1 were seen sixteen times, but could not be precisely identified due to the decondensation of heterochromatin at these regions (Fig. 3). Other chromosome bands frequently affected were 16q23 (eight times), lq44 (five times) lp36,3p24,3q27 and 10q22 (four times) and 2q37 (three times). The total number of lesions in the eight bands represented 47.5% of the chromosome lesions identified (48001).
95
TABLE
I
Frequencies Media
of chromosome Number samples
lesions and coincidence of
Number of metaphases
analyzed
RPMI-1640 Chang
Total
175 69
244
with fragile sites. Number of metaphases
Total number
Number of identified CL
with chromosome lesions (‘A/;,)
of CL/cell (%)
that coincide with FS bands (‘XS)
2201
187
199
839
(8.5) 69
(9.0) 78
(8.2)
(9.3)
50 (75.7) 27 (77. I)
256
277
77
(8.4)
(9.0
(76.2)
3040
((%I)
d Fig. 1. Partial metaphases quential Wright-G-banding.
showing
chromosome
lesions at FS-bands:
(a, c), Leishman-stained;
(b. d), se-
‘-
A
.
.. .
’
91
.: KiL-
22
!
::
I
4
t
I
0
a; ,j
d ,:
I. I.
01
‘
,
6
‘( ??) sauJosotuo~q3 s~3
: -& EL z
6q pale3!pu! ~2 spueq-g3
uwny
E
8
I
”.........“.”
‘:
El
JO uo!lnqpls!a
Es ..!
‘n
oz
u! suo!sal amosou~o~q~ snoaueluods
61
.e ‘%!A
97
Fig. 3. Partial metaphases showing chromosome lesions at lq12-q21.
To try to determine any possible influence of external factors other than culture media, the results were grouped according to age of gestation (Table II). No signiticant differences were observed among the different groups. Discussion The frequency of spontaneous chromosome lesions detected in CVS (9.1%) is statistically different (P < 0.005) from the frequency observed in lymphocytes from normal control individuals studied in our laboratory (3.4% and 2”/0in RPMI-1640 and Chang media, respectively), but is similar to the frequency of gaps and breaks in cultured control cells from first-trimester placental tissues (7.7%) [3]. On the other hand our results indicate that neither the culture medium employed nor the gestational age have a significant influence on the incidence of spontaneous chromosome lesions in CVS. TABLE II Distribution of chromosome lesions according to age of gestation Number of of samples
Number of metaphases analyzed
Number of metaphases with chromosome lesions (X0)
Total chromosome lesions (‘X,)
8-14
200
2510
IS-19
32
380
>20
8
99
219 (8.7) 22 (5.8) 9 (9.1)
237 (9.4) 24 (6.3) 9 (9.1)
Total (“h)
240”
250 (8.3)
270 (9)
Weeks of gestation
2989
‘In four cases the age of gestation was not known.
98
The chromosome localization of chromosome lesions (CL) observed in our study showed a coincidence of 76.2% with fragile site (FS) bands. If the relative length of the affected bands is taken into account it can be seen that CL are preferentially and could result from the spontaneous located in FS bands (x 2 = 430. P c 0.005) expression of FS in CV cultuies. Due to the short culture time (24 h) this could reflect an in vivo situation. However, it must be noted that a majority of CL (77%) were of the chromatid type. A total of 48 (47.5%) lesions identified clustered into eight chromosome bands; of these, 16 (15.8%) CL affected the lq12- lq21.1 region. These latter anomalies could be related to the undercondensation of heterochromatin of lqh in CVS; however, other undercondensed heterochromatin regions, such as 9h and 16h, were not especially affected by CL in CVS [lo], and sperm chromosomes do not show an accumulation of breaks at lqh, 9qh or 16qh although these regions are extremely undercondensed in these samples [4]. Abnormalities of chromosome 1 are frequently observed in solid malignancies. Brito-Babapulle and Atkin [ 1l] surveyed 343 breakpoints that led to chromosome 1 abnormalities in 218 human neoplasms and found that 49.9% were located in or near the heterochromatic regions. Breaks at lq12-q21 have been reported in choriocarcinoma cell lines [ 12,131; in one of them, a deletion affecting 1q12 was present in 50% of the analyzed metaphases. Breaks in the heterochromatic regions of chromosomes 1 and 9 were found in two of four choriocarcinoma cell lines studied by Sheppard et al. [ 121 and Habibian and Surti [3]. Centromere breaks of chromosome 1 have been observed in throphoblastic cells from complete hydatiform moles, in normal trophoblasts and in decidual cells ]31. The involvement of lql l-q21 in the chromosomal reorganizations of choriocarcinomata and the high level of spontaneous CL expression in CV cells observed in our study suggest that this region could have some special behavior in this type of tissue. The other FS with a clustering of breakpoints in our study were lq36, lq44,2q37, 3~24, 3q27, lOq22 and 16q23. None of these bands coincided with the breakpoints described by Habibiani and Surti [3] in normal trophoblasts, although these authors detected only seven breakpoints in their study. Further studies will be needed to determine whether spontaneous expression of some FS is a normal characteristic of embryonic tissues, as suggested by our current observations. Such information would provide an improved basis for assessing the occurrence of fragile sites encountered during prenatal cytogenetical diagnosis. References Fuster, C. (1987): Estudio de la fragilidad cromosomica en la especie humana. Ph.D. thesis, Universitat Autbnoma de Barcelona, Spain. Hecht, F., Tajara, E.H., Lockwood, D., Sandberg, A.A. and Hecht, B.K. (1988): New common fragile sites. Cancer Genet. Cytogenet., 33, l-9. Habibian, R. and Surti, V. (1987): Cytogenetics of trophoblasts from complete hydatidiform moles. Cancer Genet. Cytogenet., 29, 271-287. Benet, J., Fuster, C., Genesca, A., Navarro, J., Mire, R., Egozcue, J. and Templado, C. (1989): Expression of fragile sites in human sperm and lymphocyte chromosomes. Hum. Genet., 81, 239-242.
99
5
6
8
9
10
II 12 13
Abe, S., Nishida-Umehara, Ch., Tamura, T., Mikuni, C.H. and Sasaki, M. (1989): Spontaneous chromosome fragility in Band 3q21, I I pl In or I I q I3 of cultured bone marrow cells from two patients with hematologic disorders. Cancer Genet. Cytogenet., 40, 47-53. Casalone, R., Minelli, E., Portensoso, P. and Giudici, A. (1990): Clonal duplication of the Y chromosome and fra (X) in a case of epidermoid carcinoma of the esophagus. Cancer Genet. Cytogenet., 45, 269-271. Sutherland, G.R. and Ledbetter, D.H. (1989): Report of the commitee on cytogenetic markers (10th International Workshop on Human Gene Mapping). Cytogenet Cell Genet., 51, 452-458. Simoni, G., Brambati, B., Danesino, C., Rossela, F., Terzoli, G.L., Ferrari, M. and Fraccaro, M. (1983): Efficient direct chromosome analysis and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum. Genet., 63, 349-357. Perez, M.M., Miguez, L., Fuster, C., Hernandez, G. and Egozcue, J. (1990): Tecnica secuencial de bandeo cromosomico Leishman-Wright en vellosidades corionicas. Prog. Diag. Prenatal 2 (3), 145-147. Perez, M.M., Fuster, C., Mire, R., Miguez, L. and Egozcue, J. (1989): Chromosome gaps and breaks and hetercchromatin decondensation in 62 chorionic villus samples. In: Chorionic Villus Sampling and Early Prenatal Diagnosis, pp. 231-235. Editors: A. Antsaklis and C. Metaxoton. BETA Medical Arts, Athens. Brito-Babapulle, V. and Atkin, N.B. (1981): Breakpoints in chromosome I abnormalities of 218 human neoplasms. Cancer Genet. Cytogenet., 4, 215-225. Sheppard, P.M., Fisher, R.A. and Lawler, SD. (1985): Karyotypyc analysis and chromosome polymorphisms in four choriocarcinoma cell lines. Cancer Genet. Cytogenet., 16, 251-258. Surti, V. and Habibian, R. (1989): Chromosomal rearrangement in choriocarcinoma cell lines. Cancer Genet. Cytogenet., 38, 229-240.
26 (1991) 93-99
93
Elsevier Scientific Publishers Ireland Ltd. EHD 01169
Spontaneous
chromosome fragility villus cells
in chorionic
L. Miguez, C. Fuster, M.M. PCrez, R. Mir6 and J. Egozcue Departamenr
de Biologia Cellular i Fisiologia, Universitat Autbnoma de Barcelona, Barcelona (Spain)
E-08193. Bellaterra.
(Received 18 March 1991; accepted 7 June 1991)
Summary Human fragile sites are only very rarely expressed spontaneously. In this paper we report the presence of non-random spontaneous chromosome lesions (CL) in chorionic villus samples and their coincidence with fragile site (FS) bands. The average number ofCL was about 9% both in RPMI-1640 and in Chang media. To determine any possible influence of external factors other than culture media, the results were grouped according to age of gestation. No differences were observed among the different groups. A total of 101 chromosome lesions could be precisely identified by sequential Leishman Staining/Wright G-banding; 76.2% of them coincided with FS-bands. The most affected region was at lq12-lq21.1 (15.8% of total CL); other FS with a clustering of breakpoints in our study were 1~36, lq44, 2q37, 3~24, 3q27, lOq22 and 16q23. These results suggest that spontaneous expression of some FS could be a characteristic of embryonic tissues.
chromosome fragility; chromosome
lesions; embryonic tissue; chorionic villus cells
Introduction
Fragile sites on human chromosomes are weak points in the genome where nonrandom breaks or gaps can be induced by certain culture conditions. It is known that spontaneous gaps and breaks detected in control lymphocytes are concentrated at high-expression common fragile sites [ 1,2]. Correspondence to: Luz Miguez, Departament de Biologia Cellular i Fisiologia, Subunitat de Biologia Cellular, Facultat de Ciencies, Universitat Autbnoma de Barcelona, E-08 193 Bellaterra Barcelona (Spain)
0378-3782/91/$3.50 0 1991 Elsevier Scientitic Publishers Ireland Ltd. Published and Printed in Ireland
94
Spontaneous expression of fragile sites has been found in first-trimester placental tissues and molar tissues [3] in human sperm chromosomes [4], in cultured bone marrow cells from two patients with hematologic disorders [5] and in a case of epidermoid carcinoma of the esophagus [6]. In this paper we report the presence of non-random, spontaneous chromosome lesions in chorionic villus samples and their coincidence with FS accepted by the committee on cytogenetic markers [7]. Material and Methods
Chorionic villus samples were obtained from 244 cases, with gestational ages of 8-14 weeks (200) 15-19 weeks (32) or more than 20 weeks (8); in four cases the gestational age was not exactly known. Tissues samples were cultured for 24 h using a modification of the method of Simoni et al. [8]; colcemid (final concentration 0.1 &ml) was added to cultures 50 min before harvesting, and preparations made on prewarmed slides (40°C) after samples had been kept overnight at 4°C. CVS were grown in RPMI-1640 (175 cases) or Chang media (69 cases). A sequential technique Leishman-Wright G-Banding of chromosomes from chorionic villus samples to characterize chromosome lesions was used [9]. The preparations were stained with Leishman (1:4 in Leishman buffer) for 30 min, washed with distilled water and photographed. Later on, preparations, aged for 3-4 days at room temperature, were destained for 1 min each in methanol and distilled water. Immediately afterwards, preparations were incubated for 3-5 min in 2 x SSC at 65°C washed with distilled water, air dried and stained for 3 min with Wright’s (1:3 in Sorensen’s buffer, pH 6.8). Results
The frequencies of spontaneous chromosome gaps and breaks detected in our study are indicated in Table I. The average number of chromosome lesions (CL) was 9.0% in RPMI-1640 medium and 9.3% in Chang medium. Of the 199 gaps and breaks observed in the cells cultured in RPMI-1640 medium, 23.6% (471199 CL) were of the chromosome type and 76.4% (1521199) were of the chromatid type: the corresponding figures for the cells cultured in Chang medium were 21.8% (17/78 CL) and 78.2% (61/78 CL). The use of the sequential technique, Leishman staining Gbanding, allowed the precise localization of chromosome bands affected by chromosome lesions (Fig. 1). Due to the quality of CV chromosomes, only 36”/0of preparations could be successfully banded to allow the exact localization of chromosome lesions; the total number of lesions exactly identified was 101. The distribution of the CL identified was non-random (Fig. 2): 76.2% of them coincided with fragile sites (77/101 CL). Lesions at bands lq12 or q21.1 were seen sixteen times, but could not be precisely identified due to the decondensation of heterochromatin at these regions (Fig. 3). Other chromosome bands frequently affected were 16q23 (eight times), lq44 (five times) lp36,3p24,3q27 and 10q22 (four times) and 2q37 (three times). The total number of lesions in the eight bands represented 47.5% of the chromosome lesions identified (48001).
95
TABLE
I
Frequencies Media
of chromosome Number samples
lesions and coincidence of
Number of metaphases
analyzed
RPMI-1640 Chang
Total
175 69
244
with fragile sites. Number of metaphases
Total number
Number of identified CL
with chromosome lesions (‘A/;,)
of CL/cell (%)
that coincide with FS bands (‘XS)
2201
187
199
839
(8.5) 69
(9.0) 78
(8.2)
(9.3)
50 (75.7) 27 (77. I)
256
277
77
(8.4)
(9.0
(76.2)
3040
((%I)
d Fig. 1. Partial metaphases quential Wright-G-banding.
showing
chromosome
lesions at FS-bands:
(a, c), Leishman-stained;
(b. d), se-
‘-
A
.
.. .
’
91
.: KiL-
22
!
::
I
4
t
I
0
a; ,j
d ,:
I. I.
01
‘
,
6
‘( ??) sauJosotuo~q3 s~3
: -& EL z
6q pale3!pu! ~2 spueq-g3
uwny
E
8
I
”.........“.”
‘:
El
JO uo!lnqpls!a
Es ..!
‘n
oz
u! suo!sal amosou~o~q~ snoaueluods
61
.e ‘%!A
97
Fig. 3. Partial metaphases showing chromosome lesions at lq12-q21.
To try to determine any possible influence of external factors other than culture media, the results were grouped according to age of gestation (Table II). No signiticant differences were observed among the different groups. Discussion The frequency of spontaneous chromosome lesions detected in CVS (9.1%) is statistically different (P < 0.005) from the frequency observed in lymphocytes from normal control individuals studied in our laboratory (3.4% and 2”/0in RPMI-1640 and Chang media, respectively), but is similar to the frequency of gaps and breaks in cultured control cells from first-trimester placental tissues (7.7%) [3]. On the other hand our results indicate that neither the culture medium employed nor the gestational age have a significant influence on the incidence of spontaneous chromosome lesions in CVS. TABLE II Distribution of chromosome lesions according to age of gestation Number of of samples
Number of metaphases analyzed
Number of metaphases with chromosome lesions (X0)
Total chromosome lesions (‘X,)
8-14
200
2510
IS-19
32
380
>20
8
99
219 (8.7) 22 (5.8) 9 (9.1)
237 (9.4) 24 (6.3) 9 (9.1)
Total (“h)
240”
250 (8.3)
270 (9)
Weeks of gestation
2989
‘In four cases the age of gestation was not known.
98
The chromosome localization of chromosome lesions (CL) observed in our study showed a coincidence of 76.2% with fragile site (FS) bands. If the relative length of the affected bands is taken into account it can be seen that CL are preferentially and could result from the spontaneous located in FS bands (x 2 = 430. P c 0.005) expression of FS in CV cultuies. Due to the short culture time (24 h) this could reflect an in vivo situation. However, it must be noted that a majority of CL (77%) were of the chromatid type. A total of 48 (47.5%) lesions identified clustered into eight chromosome bands; of these, 16 (15.8%) CL affected the lq12- lq21.1 region. These latter anomalies could be related to the undercondensation of heterochromatin of lqh in CVS; however, other undercondensed heterochromatin regions, such as 9h and 16h, were not especially affected by CL in CVS [lo], and sperm chromosomes do not show an accumulation of breaks at lqh, 9qh or 16qh although these regions are extremely undercondensed in these samples [4]. Abnormalities of chromosome 1 are frequently observed in solid malignancies. Brito-Babapulle and Atkin [ 1l] surveyed 343 breakpoints that led to chromosome 1 abnormalities in 218 human neoplasms and found that 49.9% were located in or near the heterochromatic regions. Breaks at lq12-q21 have been reported in choriocarcinoma cell lines [ 12,131; in one of them, a deletion affecting 1q12 was present in 50% of the analyzed metaphases. Breaks in the heterochromatic regions of chromosomes 1 and 9 were found in two of four choriocarcinoma cell lines studied by Sheppard et al. [ 121 and Habibian and Surti [3]. Centromere breaks of chromosome 1 have been observed in throphoblastic cells from complete hydatiform moles, in normal trophoblasts and in decidual cells ]31. The involvement of lql l-q21 in the chromosomal reorganizations of choriocarcinomata and the high level of spontaneous CL expression in CV cells observed in our study suggest that this region could have some special behavior in this type of tissue. The other FS with a clustering of breakpoints in our study were lq36, lq44,2q37, 3~24, 3q27, lOq22 and 16q23. None of these bands coincided with the breakpoints described by Habibiani and Surti [3] in normal trophoblasts, although these authors detected only seven breakpoints in their study. Further studies will be needed to determine whether spontaneous expression of some FS is a normal characteristic of embryonic tissues, as suggested by our current observations. Such information would provide an improved basis for assessing the occurrence of fragile sites encountered during prenatal cytogenetical diagnosis. References Fuster, C. (1987): Estudio de la fragilidad cromosomica en la especie humana. Ph.D. thesis, Universitat Autbnoma de Barcelona, Spain. Hecht, F., Tajara, E.H., Lockwood, D., Sandberg, A.A. and Hecht, B.K. (1988): New common fragile sites. Cancer Genet. Cytogenet., 33, l-9. Habibian, R. and Surti, V. (1987): Cytogenetics of trophoblasts from complete hydatidiform moles. Cancer Genet. Cytogenet., 29, 271-287. Benet, J., Fuster, C., Genesca, A., Navarro, J., Mire, R., Egozcue, J. and Templado, C. (1989): Expression of fragile sites in human sperm and lymphocyte chromosomes. Hum. Genet., 81, 239-242.
99
5
6
8
9
10
II 12 13
Abe, S., Nishida-Umehara, Ch., Tamura, T., Mikuni, C.H. and Sasaki, M. (1989): Spontaneous chromosome fragility in Band 3q21, I I pl In or I I q I3 of cultured bone marrow cells from two patients with hematologic disorders. Cancer Genet. Cytogenet., 40, 47-53. Casalone, R., Minelli, E., Portensoso, P. and Giudici, A. (1990): Clonal duplication of the Y chromosome and fra (X) in a case of epidermoid carcinoma of the esophagus. Cancer Genet. Cytogenet., 45, 269-271. Sutherland, G.R. and Ledbetter, D.H. (1989): Report of the commitee on cytogenetic markers (10th International Workshop on Human Gene Mapping). Cytogenet Cell Genet., 51, 452-458. Simoni, G., Brambati, B., Danesino, C., Rossela, F., Terzoli, G.L., Ferrari, M. and Fraccaro, M. (1983): Efficient direct chromosome analysis and enzyme determinations from chorionic villi samples in the first trimester of pregnancy. Hum. Genet., 63, 349-357. Perez, M.M., Miguez, L., Fuster, C., Hernandez, G. and Egozcue, J. (1990): Tecnica secuencial de bandeo cromosomico Leishman-Wright en vellosidades corionicas. Prog. Diag. Prenatal 2 (3), 145-147. Perez, M.M., Fuster, C., Mire, R., Miguez, L. and Egozcue, J. (1989): Chromosome gaps and breaks and hetercchromatin decondensation in 62 chorionic villus samples. In: Chorionic Villus Sampling and Early Prenatal Diagnosis, pp. 231-235. Editors: A. Antsaklis and C. Metaxoton. BETA Medical Arts, Athens. Brito-Babapulle, V. and Atkin, N.B. (1981): Breakpoints in chromosome I abnormalities of 218 human neoplasms. Cancer Genet. Cytogenet., 4, 215-225. Sheppard, P.M., Fisher, R.A. and Lawler, SD. (1985): Karyotypyc analysis and chromosome polymorphisms in four choriocarcinoma cell lines. Cancer Genet. Cytogenet., 16, 251-258. Surti, V. and Habibian, R. (1989): Chromosomal rearrangement in choriocarcinoma cell lines. Cancer Genet. Cytogenet., 38, 229-240.