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A new human cell line derived from a poorly differentiated endometrial adenocarcinoma
GYNECOLOGIC ONCOLOGY
13 50-57 (1982)
A New Human Cell Line Derived from a Poorly Differentiated Endometrial Adenocarcinoma DAVID GAL,’
J. PETER FORNEY, V. G. DEV, AND JOHN C. PORTER
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology and the Division of Cytogenetics of the Department of Pathology, University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235 Accepted June 25, 1981
Establishment and characterization of an endometrial adenocarcinoma cell line are described. The tumor cells, designated AC-258, originated from a patient with a poorly differentiated adenocarcinoma of the endometrium. These cells have been passed in culture more than 102 times and have maintained their morphological and chromosomal integrity in a fashion suggestive of monocloning. Cells from passages 1, 58, and 102 were aneuploid. The average number of chromosomes per cell was 64.8, and 8-17 marker chromosomes per cell were identified and described. Histological evaluation of tumor explants from 3 passages grown in nude (athymic) mice revealed morphologic identity. The doubling time of AC-258 was 22 hr. No detectable estrogen- or progesterone-binding proteins were found. The AC-258 cell line provides researchers with another tool with which to investigate biochemical and biological properties of human cancer cells.
INTRODUCTION Techniques that enable investigators to establish and maintain in culture cells derived from human neoplastic tissues have proven to be highly useful in the study of malignant cells. Although the full potential of studies involving such cells has probably not been realized, it can be envisioned that cancer cells derived from gynecologic malignancies will aid in studies on cellular sensitivity to cytotoxic agents. Neoplastic cells in culture may also provide a source of tumorassociated antigens. Purified antigens could be highly useful in the production of monoclonal antibodies. Such antibodies could be of considerable utility in both diagnosis and therapy of genital tract cancer. Pure cell cultures derived from gynecologic neoplasms could also be of use in the study of cytogenetics, hormone production, hormone receptor sites, cell membrane research, and in the search for understanding of the basis underlying the transformation of normal cells to malignant cells. ’ To whom correspondence and requests for reprints should be addressed at: Department of Obstetrics and Gynecology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Tex. 75235. 50 0090-8258/82/010050-08$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
51
Investigators interested in neoplastic cells from human gynecologic tissues have succeeded in establishing in culture a variety of cancer cell lines. Such cell lines have been derived from epidermoid cancer of the cervix [I], epidermoid cancer of the vagina [1], adenocarcinoma of the ovary [2], and others [3-71. Cell lines derived from human endometrial adenocarcinomas have proven to be difficult to establish, and, heretofore, only five such lines have been described [3-71. Except for one [4], all were derived from well-differentiated neoplasms. In the present report, a cell line derived from a poorly differentiated endometrial adenocarcinoma is described with respect to its establishment, maintenance, morphology, growth kinetics, chromosomal content, and sex steroid receptors. MATERIALS
AND METHODS
History of the Donor of Endometrial Adenocarcinoma Cells The donor of the tissue used in these studies was a 55year-old nulligravid, postmenopausal, obese, black woman, who, following cervical dilatation and uterine curettage, was found to have poorly differentiated adenocarcinoma of the endometrium, Stage IB, Grade 3. At celiotomy, the uterine wall was seen to have been penetrated by the malignancy, and metastases were present in the omentum and para-aortic lymph nodes. The ovaries were not involved with the tumor. A hysterectomy, bilateral salpingo-oophorectomy, and omental biopsy were performed. Despite three courses of cytotoxic chemotherapy and daily oral administration of a synthetic progestin, megestrol acetate, the residual malignancy continued to grow and metastasize. The patient’s clinical condition progressively deteriorated, and she died 4 months after celiotomy. Neoplastic tissue was obtained from the omental metastasis in accordance with the Donors Anatomical Gift Act of the State of Texas after obtaining consent in writing from the patient employing a consent form and protocol approved by the Human Research Review Committee of the University of Texas Health Science Center at Dallas. This tissue was utilized to establish the endometrial adenocarcinoma cell line identified as AC-258. The metastasis was histologically identical to the large, polypoid primary tumor present within the endometrial cavity (Figs. lA, B). Culture Technique Tissue from the metastatic tumor nodule was minced with scissors under aseptic conditions and placed in T-25 plastic culture flasks containing the following types of media enriched with 10% fetal calf serum: Waymouth MB7521, RPMI, McCoy’s Sa, Ham’s, and Dulbecco’s minimum essential medium. All cultures were incubated at 37°C and the media were changed frequently. Cells were passed when confluency was achieved, and following the third passage, contaminating fibroblasts were eliminated by differential sedimentation. This technique involved incubation of cultured cells for 30 min at 37°C after which the culture medium and unattached cells were transferred to another flask. Fibroblasts were the first cells to attach to the flask, and after this procedure was repeated five times, contaminating fibroblasts were reduced to an insignificant number.
52
GAL ET AL.
F'IG. 1. Photomicrographs (x 250): (A) histological section of the primary tumor obtained from the endometrial cavity: (B) omental metastasis, which served as the source of endometrial aclenocart:inoma cells; (C) tumor grown in a nude male mouse after inoculation with cells from passage I; aInd (D) tumor grown in a nude male mouse after inoculation with cells from passage 42.
ENDOMETRIAL
CANCER CELLS IN CULTURE
53
Heterotrunsplantation. One million tumor cells obtained from passages 7 and 42 were injected subcutaneously into athymic, nude mice. When tumor nodules were 5-10 mm in diameter, the animals were killed, the tumors excised, and the tumorous tissue was prepared for light microscopic examination. Growth kinetics. Growth studies were performed on AC-258 cells obtained from passage 51. Cells (3 x 105)were placed in Petri dishes. At 24-hr intervals for 6 days, cells were removed from representative dishes and analyzed for protein by the method of Lowry et al. [S] and for DNA according to the procedure of Burton [9]. Chromosomul analysis. Suspensions of preconfluent tumor cells from passages 1, 58, and 102 were incubated with colcemid (1 mg x ml-‘) for 30 min and then pelleted by centrifugation at 25Og for 7 min. After centrifugation, the cells were resuspended in a hypotonic solution of KC1 (0.5%) and incubated for 10 min at room temperature. The cells were then fixed with a methanol:acetic acid solution (3:l) for 60 min. Quinecrine fluorescence banding was performed according to the method of Dev et al. [lo]. Chromosomes were analyzed by means of fluorescent microscopy. Progesterone and 17f3-Estrudiol Receptor Analysis Preconfluent and confluent tumor cells from passage 46 were analyzed for the presence of estrogen and progesterone receptors. Prior to assay, AC-258 cells were incubated for 2 days with Waymouth’s MB752-1 medium containing 10% charcoal-stripped fetal calf serum. The cells were then washed three times with phosphate buffer (0.15 M; pH 7.4) at 2”C, scraped from the dishes, and homogenized in a buffer solution (pH 7.4) containing Tris (10 mM), EDTA (1.5 mM), and DTT (1 mM) at 2°C using a Teflon-glass homogenizer. The homogenate was centrifuged (100,OOOg)for 60 min, and the supernatant fluid was analyzed for progesterone (P) and 17B-estradiol (EJ receptors. One-half of the supernatant fluid (cytosol) fraction was incubated with 0.5% Norit A, 0.05% dextran T,, for 60 min at 2°C to remove receptor-bound steroids. P binding was measured in both charcoal-treated and nontreated fractions. Each fraction was diluted to 4 mg protein x ml-’ and incubated with 5 nM [2,4,6,7-jH]P with or without 5 M nonradiolabeled P or cortisol. One set of samples was incubated for 60 min at 0°C and then analyzed by means of sucrose density centrifugation as described by Mendelson et ul. [ 111. The other set of samples was incubated at 0°C for 16 hr, after which the bound [‘HIP was separated from free steroid using dextran-coated charcoal [ 1I]. Estrogen receptors were measured using the same techniques, except that the charcoal-treated and nontreated fractions were incubated in 5 nM [2,4,6,7-3H]E2 in the presence or absence of 5 M nonradiolabeled Ez or diethylstilbesterol (DES). Materials Culture media and fetal calf serum were obtained from Grand Island Biological Company (Grand Island, N.Y.). All culture flasks were obtained from Falcon Plastics (Cockeysville, Md.). [2,4,6,7-3H]P and [2,4,6,7-‘H]E2 were purchased from New England Nuclear Corporation (Boston, Mass.).
54
GAL ET AL.
RESULTS Establishment of Tumor Cell Line After 48 hr, tumor cells from the minced tissue attached to the bottom of several flasks. Cells grew in each of the media tested but consistently grew best in Waymouth’s MB752-1, which was used exclusively during purification and subsequent maintenance. Cytology Adenocarcinoma cells, designated AC-258, appeared in culture as large, multinucleated polygonal cells with marked cytoplasmic vacuolization. Cell membranes were indistinct, and no glandular architecture was recognized. The cells grew in monolayer until confluency was achieved, whereupon the growth rate decreased, but the cells continued to grow in a multilayer fashion. Heterotransplantation AC-258 cells were injected subcutaneously into the subscapular region of nude (athymic) mice after passages 7 and 42. Eighty percent of injected male mice but no female mice developed a tumor at the site of injection. Three weeks after inoculation, the tumors attained diameters of 5-10 mm. Tumors from these passages, when examined histologically, were found to consist of cells resembling the original tumor (Figs. IB-D). Growth Kinetics The mean protein content of AC-258 cells from passage 51 was 5 16 pg per cell; the mean DNA content was 14.1 pg per cell. During the phase of maximal growth, the doubling time for AC-258 cells was 22 hr (Fig. 2). 1
70
3-
0
1 48
I 96
144
TIME (l-l) FIG. 2. Growth kinetics of AC-258 from passage 51. The protein content (O), DNA content (A), and number of cells (0) per 60-mm petri dish as a function of time in culture. The details of this experiment are described under Materials and Methods. The data presented are the average of values obtained using cells from triplicate dishes.
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
55
Chromosome Analysis Twenty well-spread cells in metaphase from passages 1, 58, and 102 were analyzed. In all parameters studied, the chromosomes of cells of each passage were remarkably similar. The number of chromosomes per cell ranged from 58 to 80, with a mean of 64.8 (Table I). The karyotype of a representative cell is shown in Fig. 3. Autosomal trisomy was common, but in every cell that was analyzed, only two X chromosomes were present. Chromosomes 9, 16, and 17 were not identified; however, portions of these chromosomes were included within rearranged marker chromosomes. The number of marker chromosomes in each of the 20 cell preparations varied from 8 to 17. Analysis of these marker chromosomes utilizing Quinecrine banding revealed the following: Marker 1 is the result of junction between chromosome 9 and chromosomal fragment lq; marker 2 is derived from chromosome 8; marker 3 is the chromosomal fragment lp; marker 4 is a result of junction between chromosome 16 and chromosomal fragment llq; marker 5 is derived from chromosomes 15 and 17; marker 7 is a result of centric fusion of chromosomes 13 and 21; marker 8 is derived from chromosome 17; marker 9 is derived from chromosomal fragment lq; marker 13 is derived from chromosomal fragment 3q. The source of markers 10, 11, and 12 could not be ascertained. Sex Steroid Analysis Sucrose density gradient centrifugation analysis of the cytosol from either confluent or preconfluent cells incubated with [‘HIP or [jH]E? revealed no detectable P- or E,-binding proteins. Furthermore, no displaceable P or Ez binding was formed in cytosolic fractions when incubated with [‘HIP or [3H]Ez in the presence or absence of lOOO-foldexcess of nonradiolabeled steroids when dextran-coated charcoal was used to separate bound from free [‘HIP or [‘H]E?. DISCUSSION
Cell line AC-258 is the sixth successfully established endometrial adenocarcinema cell line reported. The neoplasm was morphologically anaplastic and brought about the death of the explant donor within a few months of diagnosis. The doubling time of AC-258 cells was 22 hr, and this time is less than that TABLE 1 FREQUENCY
DISTRIBUTION
OF
AC-258 NUMBER
Number of chromosomes per cell 55-60 61-65 66-70 71-75 76-80
CELLS FROM PASSAGES I, 58, AND OF CHROMOSOMES PER CELL
102 WITH
RESPECT TO
Number of cells Passage I
Passage 58
Passage 102
7 5 5 2 I
6 6 4 3 1
5 7 4 2 2
56
GAL ET AL.
FIG. 3. Karyotype of a representative aneuploid AC-258 cell after Quinecrine fluorescence banding. Marker chromosomes are numbered from Ml through Ml3. See Results for description of marker chromosomes.
reported for other endometrial adenocarcinoma cells [3-71. The growth of the tumor in vitro appeared to mimic its in viva virulence, possibly facilitating the establishment of the cell line. Cytogenetic analysis of AC-258 cells revealed chromosomal aneuploidy with an average number of 64.8 chromosomes per cell. Numerous marker chromosomes were found in every cell examined, and similar markers were found in cells from passages 1, 58, and 102. This finding is suggestive that a monoclonal cell line had been established. Previously reported cell lines derived from welldifferentiated or moderately well-differentiated endometrial adenocarcinoma consisted of diploid or tetraploid cells with only a few marker chromosomes [3,5,7]. The absence of P or E, receptors may reflect the undifferentiated nature of the tumor and could explain the patient’s lack of response to progestin therapy. Of interest is the failure of tumor growth in female athymic mice, and the basis of this curious finding is presently being explored. AC-258 cells represent the sixth endometrial adenocarcinoma cell line suc-
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
57
cessfully established and maintained in tissue culture. The cell line is unique in several respects and is presently being used for investigative studies to explore the behavior of neoplastic cells. ACKNOWLEDGMENTS We thank Janette Tuckey, Gloria Cho, Mary Moore, and Patricia Hemsell for skilled technical assistance, and Dr. Carole Mendelson from the Department of Biochemistry for the analysis of steroid hormone receptors. The editorial assistance of Anita Crockett is gratefully acknowledged.
REFERENCES I. Porter, J. C., Nalick, R. H., Vellios, F., Neaves, W. B., and MacDonald, P. C. New tissue culture cell lines derived from human squamous cell carcinoma of the cervix and vagina: Squamous cells in tissue culture, Amer. J. O&star. Gynecol. 130, 487-496 (1978). 2. DiSaia, P. J., Morrow, M., Kanabus, J., Piechal, W., and Townsend, D. E. Two new tissue culture lines from ovarian cancer, Gynecol. Oncol. 3, 215-219 (1975). 3. Kuramoto, H. Studies of the growth and cytogenetic properties of human endometrial adenocarcinoma in culture and its development into an established line, Acrcr Ohsret. Gy~uecol. Japan.
19, 47-58 (1972).
4. Merenda, C., Sordat, B., Moth, J. P.. and Carrel, S. Human endometrial carcinomas serially transplanted in nude mice and established in continuous cell lines, Int. J. Cancer. 16, 559-570 (1975). 5. Kuramoto, H., and Hamono, M. Establishment and characterization of the cell line of a human endometrial adenocarcinoma, Eur. J. Cancer 13, 253-259 (1977). 6. Ishiwata, S., Nozawa, S., Inoue, T., and Okumura. H. Development and characterization of established cell lines from primary and metastatic regions of human endometrial adenocarcinema, Cancer Res. 37, 1777-1785 (1977). 7. Gorodecki, J., Mostel, R., Ladda. R. L., Ward, S. P., Geder, L., and Rapp, F. Establishment and characterization of a new endometrial cancer cell line (SCRC-I), Amer. J. Ohsret. Gynccol. 13.5, 671-679 (1979).
8. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall. R. J. Protein measurement with the Folin phenol reagent, J. Eiol. Chrm. 193, 265-275 (1951). 9. Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid, Biochem. J. 62, 315-326 (1956). IO. Dev, V. G., Miller, D. A., Miller, 0. J., Marshall, J. T., Jr., and Hsu, T. C. Quinacrine fluorescence of Mus cmicolor chromosomes. Bright centromeric heterochromatin, Exp. Cell Res. 79, 475-479 (1973). I I. Mendelson, C. R., MacDonald, P. C., and Johnston, J. M. Estrogen binding in human fetal lung tissue cytosol, Endocrinology 106, 368-374 (1980). 12. MC&ire, W. L., and Delagarza, M. Improved sensitivity in the measurement of estrogen receptor in human breast cancer, /. C/in. Endocrinol. Mefahol. 37, 986-989 (1973).
13 50-57 (1982)
A New Human Cell Line Derived from a Poorly Differentiated Endometrial Adenocarcinoma DAVID GAL,’
J. PETER FORNEY, V. G. DEV, AND JOHN C. PORTER
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology and the Division of Cytogenetics of the Department of Pathology, University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235 Accepted June 25, 1981
Establishment and characterization of an endometrial adenocarcinoma cell line are described. The tumor cells, designated AC-258, originated from a patient with a poorly differentiated adenocarcinoma of the endometrium. These cells have been passed in culture more than 102 times and have maintained their morphological and chromosomal integrity in a fashion suggestive of monocloning. Cells from passages 1, 58, and 102 were aneuploid. The average number of chromosomes per cell was 64.8, and 8-17 marker chromosomes per cell were identified and described. Histological evaluation of tumor explants from 3 passages grown in nude (athymic) mice revealed morphologic identity. The doubling time of AC-258 was 22 hr. No detectable estrogen- or progesterone-binding proteins were found. The AC-258 cell line provides researchers with another tool with which to investigate biochemical and biological properties of human cancer cells.
INTRODUCTION Techniques that enable investigators to establish and maintain in culture cells derived from human neoplastic tissues have proven to be highly useful in the study of malignant cells. Although the full potential of studies involving such cells has probably not been realized, it can be envisioned that cancer cells derived from gynecologic malignancies will aid in studies on cellular sensitivity to cytotoxic agents. Neoplastic cells in culture may also provide a source of tumorassociated antigens. Purified antigens could be highly useful in the production of monoclonal antibodies. Such antibodies could be of considerable utility in both diagnosis and therapy of genital tract cancer. Pure cell cultures derived from gynecologic neoplasms could also be of use in the study of cytogenetics, hormone production, hormone receptor sites, cell membrane research, and in the search for understanding of the basis underlying the transformation of normal cells to malignant cells. ’ To whom correspondence and requests for reprints should be addressed at: Department of Obstetrics and Gynecology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Tex. 75235. 50 0090-8258/82/010050-08$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
51
Investigators interested in neoplastic cells from human gynecologic tissues have succeeded in establishing in culture a variety of cancer cell lines. Such cell lines have been derived from epidermoid cancer of the cervix [I], epidermoid cancer of the vagina [1], adenocarcinoma of the ovary [2], and others [3-71. Cell lines derived from human endometrial adenocarcinomas have proven to be difficult to establish, and, heretofore, only five such lines have been described [3-71. Except for one [4], all were derived from well-differentiated neoplasms. In the present report, a cell line derived from a poorly differentiated endometrial adenocarcinoma is described with respect to its establishment, maintenance, morphology, growth kinetics, chromosomal content, and sex steroid receptors. MATERIALS
AND METHODS
History of the Donor of Endometrial Adenocarcinoma Cells The donor of the tissue used in these studies was a 55year-old nulligravid, postmenopausal, obese, black woman, who, following cervical dilatation and uterine curettage, was found to have poorly differentiated adenocarcinoma of the endometrium, Stage IB, Grade 3. At celiotomy, the uterine wall was seen to have been penetrated by the malignancy, and metastases were present in the omentum and para-aortic lymph nodes. The ovaries were not involved with the tumor. A hysterectomy, bilateral salpingo-oophorectomy, and omental biopsy were performed. Despite three courses of cytotoxic chemotherapy and daily oral administration of a synthetic progestin, megestrol acetate, the residual malignancy continued to grow and metastasize. The patient’s clinical condition progressively deteriorated, and she died 4 months after celiotomy. Neoplastic tissue was obtained from the omental metastasis in accordance with the Donors Anatomical Gift Act of the State of Texas after obtaining consent in writing from the patient employing a consent form and protocol approved by the Human Research Review Committee of the University of Texas Health Science Center at Dallas. This tissue was utilized to establish the endometrial adenocarcinoma cell line identified as AC-258. The metastasis was histologically identical to the large, polypoid primary tumor present within the endometrial cavity (Figs. lA, B). Culture Technique Tissue from the metastatic tumor nodule was minced with scissors under aseptic conditions and placed in T-25 plastic culture flasks containing the following types of media enriched with 10% fetal calf serum: Waymouth MB7521, RPMI, McCoy’s Sa, Ham’s, and Dulbecco’s minimum essential medium. All cultures were incubated at 37°C and the media were changed frequently. Cells were passed when confluency was achieved, and following the third passage, contaminating fibroblasts were eliminated by differential sedimentation. This technique involved incubation of cultured cells for 30 min at 37°C after which the culture medium and unattached cells were transferred to another flask. Fibroblasts were the first cells to attach to the flask, and after this procedure was repeated five times, contaminating fibroblasts were reduced to an insignificant number.
52
GAL ET AL.
F'IG. 1. Photomicrographs (x 250): (A) histological section of the primary tumor obtained from the endometrial cavity: (B) omental metastasis, which served as the source of endometrial aclenocart:inoma cells; (C) tumor grown in a nude male mouse after inoculation with cells from passage I; aInd (D) tumor grown in a nude male mouse after inoculation with cells from passage 42.
ENDOMETRIAL
CANCER CELLS IN CULTURE
53
Heterotrunsplantation. One million tumor cells obtained from passages 7 and 42 were injected subcutaneously into athymic, nude mice. When tumor nodules were 5-10 mm in diameter, the animals were killed, the tumors excised, and the tumorous tissue was prepared for light microscopic examination. Growth kinetics. Growth studies were performed on AC-258 cells obtained from passage 51. Cells (3 x 105)were placed in Petri dishes. At 24-hr intervals for 6 days, cells were removed from representative dishes and analyzed for protein by the method of Lowry et al. [S] and for DNA according to the procedure of Burton [9]. Chromosomul analysis. Suspensions of preconfluent tumor cells from passages 1, 58, and 102 were incubated with colcemid (1 mg x ml-‘) for 30 min and then pelleted by centrifugation at 25Og for 7 min. After centrifugation, the cells were resuspended in a hypotonic solution of KC1 (0.5%) and incubated for 10 min at room temperature. The cells were then fixed with a methanol:acetic acid solution (3:l) for 60 min. Quinecrine fluorescence banding was performed according to the method of Dev et al. [lo]. Chromosomes were analyzed by means of fluorescent microscopy. Progesterone and 17f3-Estrudiol Receptor Analysis Preconfluent and confluent tumor cells from passage 46 were analyzed for the presence of estrogen and progesterone receptors. Prior to assay, AC-258 cells were incubated for 2 days with Waymouth’s MB752-1 medium containing 10% charcoal-stripped fetal calf serum. The cells were then washed three times with phosphate buffer (0.15 M; pH 7.4) at 2”C, scraped from the dishes, and homogenized in a buffer solution (pH 7.4) containing Tris (10 mM), EDTA (1.5 mM), and DTT (1 mM) at 2°C using a Teflon-glass homogenizer. The homogenate was centrifuged (100,OOOg)for 60 min, and the supernatant fluid was analyzed for progesterone (P) and 17B-estradiol (EJ receptors. One-half of the supernatant fluid (cytosol) fraction was incubated with 0.5% Norit A, 0.05% dextran T,, for 60 min at 2°C to remove receptor-bound steroids. P binding was measured in both charcoal-treated and nontreated fractions. Each fraction was diluted to 4 mg protein x ml-’ and incubated with 5 nM [2,4,6,7-jH]P with or without 5 M nonradiolabeled P or cortisol. One set of samples was incubated for 60 min at 0°C and then analyzed by means of sucrose density centrifugation as described by Mendelson et ul. [ 111. The other set of samples was incubated at 0°C for 16 hr, after which the bound [‘HIP was separated from free steroid using dextran-coated charcoal [ 1I]. Estrogen receptors were measured using the same techniques, except that the charcoal-treated and nontreated fractions were incubated in 5 nM [2,4,6,7-3H]E2 in the presence or absence of 5 M nonradiolabeled Ez or diethylstilbesterol (DES). Materials Culture media and fetal calf serum were obtained from Grand Island Biological Company (Grand Island, N.Y.). All culture flasks were obtained from Falcon Plastics (Cockeysville, Md.). [2,4,6,7-3H]P and [2,4,6,7-‘H]E2 were purchased from New England Nuclear Corporation (Boston, Mass.).
54
GAL ET AL.
RESULTS Establishment of Tumor Cell Line After 48 hr, tumor cells from the minced tissue attached to the bottom of several flasks. Cells grew in each of the media tested but consistently grew best in Waymouth’s MB752-1, which was used exclusively during purification and subsequent maintenance. Cytology Adenocarcinoma cells, designated AC-258, appeared in culture as large, multinucleated polygonal cells with marked cytoplasmic vacuolization. Cell membranes were indistinct, and no glandular architecture was recognized. The cells grew in monolayer until confluency was achieved, whereupon the growth rate decreased, but the cells continued to grow in a multilayer fashion. Heterotransplantation AC-258 cells were injected subcutaneously into the subscapular region of nude (athymic) mice after passages 7 and 42. Eighty percent of injected male mice but no female mice developed a tumor at the site of injection. Three weeks after inoculation, the tumors attained diameters of 5-10 mm. Tumors from these passages, when examined histologically, were found to consist of cells resembling the original tumor (Figs. IB-D). Growth Kinetics The mean protein content of AC-258 cells from passage 51 was 5 16 pg per cell; the mean DNA content was 14.1 pg per cell. During the phase of maximal growth, the doubling time for AC-258 cells was 22 hr (Fig. 2). 1
70
3-
0
1 48
I 96
144
TIME (l-l) FIG. 2. Growth kinetics of AC-258 from passage 51. The protein content (O), DNA content (A), and number of cells (0) per 60-mm petri dish as a function of time in culture. The details of this experiment are described under Materials and Methods. The data presented are the average of values obtained using cells from triplicate dishes.
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
55
Chromosome Analysis Twenty well-spread cells in metaphase from passages 1, 58, and 102 were analyzed. In all parameters studied, the chromosomes of cells of each passage were remarkably similar. The number of chromosomes per cell ranged from 58 to 80, with a mean of 64.8 (Table I). The karyotype of a representative cell is shown in Fig. 3. Autosomal trisomy was common, but in every cell that was analyzed, only two X chromosomes were present. Chromosomes 9, 16, and 17 were not identified; however, portions of these chromosomes were included within rearranged marker chromosomes. The number of marker chromosomes in each of the 20 cell preparations varied from 8 to 17. Analysis of these marker chromosomes utilizing Quinecrine banding revealed the following: Marker 1 is the result of junction between chromosome 9 and chromosomal fragment lq; marker 2 is derived from chromosome 8; marker 3 is the chromosomal fragment lp; marker 4 is a result of junction between chromosome 16 and chromosomal fragment llq; marker 5 is derived from chromosomes 15 and 17; marker 7 is a result of centric fusion of chromosomes 13 and 21; marker 8 is derived from chromosome 17; marker 9 is derived from chromosomal fragment lq; marker 13 is derived from chromosomal fragment 3q. The source of markers 10, 11, and 12 could not be ascertained. Sex Steroid Analysis Sucrose density gradient centrifugation analysis of the cytosol from either confluent or preconfluent cells incubated with [‘HIP or [jH]E? revealed no detectable P- or E,-binding proteins. Furthermore, no displaceable P or Ez binding was formed in cytosolic fractions when incubated with [‘HIP or [3H]Ez in the presence or absence of lOOO-foldexcess of nonradiolabeled steroids when dextran-coated charcoal was used to separate bound from free [‘HIP or [‘H]E?. DISCUSSION
Cell line AC-258 is the sixth successfully established endometrial adenocarcinema cell line reported. The neoplasm was morphologically anaplastic and brought about the death of the explant donor within a few months of diagnosis. The doubling time of AC-258 cells was 22 hr, and this time is less than that TABLE 1 FREQUENCY
DISTRIBUTION
OF
AC-258 NUMBER
Number of chromosomes per cell 55-60 61-65 66-70 71-75 76-80
CELLS FROM PASSAGES I, 58, AND OF CHROMOSOMES PER CELL
102 WITH
RESPECT TO
Number of cells Passage I
Passage 58
Passage 102
7 5 5 2 I
6 6 4 3 1
5 7 4 2 2
56
GAL ET AL.
FIG. 3. Karyotype of a representative aneuploid AC-258 cell after Quinecrine fluorescence banding. Marker chromosomes are numbered from Ml through Ml3. See Results for description of marker chromosomes.
reported for other endometrial adenocarcinoma cells [3-71. The growth of the tumor in vitro appeared to mimic its in viva virulence, possibly facilitating the establishment of the cell line. Cytogenetic analysis of AC-258 cells revealed chromosomal aneuploidy with an average number of 64.8 chromosomes per cell. Numerous marker chromosomes were found in every cell examined, and similar markers were found in cells from passages 1, 58, and 102. This finding is suggestive that a monoclonal cell line had been established. Previously reported cell lines derived from welldifferentiated or moderately well-differentiated endometrial adenocarcinoma consisted of diploid or tetraploid cells with only a few marker chromosomes [3,5,7]. The absence of P or E, receptors may reflect the undifferentiated nature of the tumor and could explain the patient’s lack of response to progestin therapy. Of interest is the failure of tumor growth in female athymic mice, and the basis of this curious finding is presently being explored. AC-258 cells represent the sixth endometrial adenocarcinoma cell line suc-
ENDOMETRIAL
CANCER
CELLS
IN CULTURE
57
cessfully established and maintained in tissue culture. The cell line is unique in several respects and is presently being used for investigative studies to explore the behavior of neoplastic cells. ACKNOWLEDGMENTS We thank Janette Tuckey, Gloria Cho, Mary Moore, and Patricia Hemsell for skilled technical assistance, and Dr. Carole Mendelson from the Department of Biochemistry for the analysis of steroid hormone receptors. The editorial assistance of Anita Crockett is gratefully acknowledged.
REFERENCES I. Porter, J. C., Nalick, R. H., Vellios, F., Neaves, W. B., and MacDonald, P. C. New tissue culture cell lines derived from human squamous cell carcinoma of the cervix and vagina: Squamous cells in tissue culture, Amer. J. O&star. Gynecol. 130, 487-496 (1978). 2. DiSaia, P. J., Morrow, M., Kanabus, J., Piechal, W., and Townsend, D. E. Two new tissue culture lines from ovarian cancer, Gynecol. Oncol. 3, 215-219 (1975). 3. Kuramoto, H. Studies of the growth and cytogenetic properties of human endometrial adenocarcinoma in culture and its development into an established line, Acrcr Ohsret. Gy~uecol. Japan.
19, 47-58 (1972).
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