Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1067-1071, March 1977 Cell Biology
Establishment and characterization of a strain of human adrenal tumor cells that secrete estrogen (Fang-8 clonal cell line/epithelioid cells)
VICTOR S. FANG Department of Medicine, The University of Chicago, Chicago, Illinois 60637
Communicated by Elwood V. Jensen, December 3, 1976
We report the establishment of a new cell line, ABSTRACT designated Fang-8, which originated from a human adrenal adenocarcinoma. It has been continuously propagated during the past 20 months. The cells exhibit an epithelioid morphology, resembling the cell structure of the zona reticularis when viewed by electron microscopy. Since passage 12, the cells showed a characteristic ring-forming property in culture. The cells produce neither testosterone, mineralocorticosteroids, nor glucocorticosteroids; instead, they possess the unique function of estrogen production. Karyotypic analysis revealed a 100% aneuploid, unstable karyotype in the hyperdiploid stem line. Identification of the Y chromosome was uncertain. In view of these characteristics, Fang-8 represents a new human cell line, unlikely to be contaminated with HeLa strains, and different from another cell line derived from human adrenal cortex adenocarcinoma, namely SW-13 (ATCC CCL 105).
Among the many mammalian cell lines deposited with the American Type Culture Collection (ATCC) (1), very few have maintained some unique biochemical function which can be utilized for further characterization of the cells in an in vitro system. Cell lines of endocrine tissue origin, by retaining hormonal production, provide an ideal model in this respect. However, due to either a phenotypic change or inadequate environmental conditions, the tissue-specific function could easily disappear during serial propagation of the cultured animal cells. In order to overcome these disadvantages, Buonassisi et al. (2) developed a method of alternate culture and animal passage, and consequently, several functioning rodent endocrine cell lines were successfully established (3). Obviously, such a technique cannot be used to establish human cell lines. Of the 53 human cell lines which have so far been deposited with ATCC (1), only one line, SW-13 (CCL 105) was derived from human adrenal cortex adenocarcinoma (4); its hormonal production was not studied. Another interesting cell line, BeWo (CCL 98), derived from choriocarcinoma (5), maintained hormone-synthesizing functions, and produced human chorionic gonadotropin and estrogens (6). During the past 2 years, I attempted to develop stable human cell lines from various neoplastic tissues which were known to produce hormones. In the present communication, I report one such clonal strain of human cells that has been propagated by serial subcultures in my laboratory and continuously produces estrogen. MATERIALS AND METHODS The cells were obtained from a patient with the following history: A.W., Jr., a black male, father of three children, noticed diminished libido, impotency, and mammary gland enlargement since 1972. He did not seek further medical treatment until May 31, 1973 when he came to the emergency room of Billings Hospital, and complained of acute abdominal pain. By exploratory laparotomy, a ruptured tumor of the right adrenal gland with large retroperitoneal hemorrhage and abscess was discovered. Histologic examination revealed an adrenal ade1067
nocarcinoma with hematoma (Fig. 1A). His postoperative serum levels of estrogen as measured by radioimmunoassay technique ranged from 90 to 240 pg/ml (normal male value in our laboratory is 50 + 15 pg/ml, mean L SD). Values for the other adrenal hormones were within normal limits. Beginning in August, 1973, the patient was treated with 5 g of mitotane, daily, but his serum estrogen levels were progressively elevated. In January, 1974 he developed hypertension. In August, 1974 hepatic metastases were detected. On February 17, 1975, a palliative gastrojejunostomy was performed to relieve gastric outlet obstruction by tumor compression. A tumor mass (4 X 4 cm) at right side was removed for histologic examination which revealed adrenal carcinoma (Fig. 1B). A small piece of the same tumor mass was cultured. The patient died of renal failure on April 15, 1975, at age 30. He suffered a metastatic feminizing adrenocortical adenocarcinoma, a relatively rare disease with poor prognosis (for a review see ref. 7). Culture Media. The medium used for the primary culture was F-10 synthetic culture medium (8) supplemented with 20% or 2.5% fetal calf serum. The medium used for serial subcultures was Matalon modified Eagle's medium (9), supplemented with 10% each of calf and fetal calf sera. All media were purchased from Grand Island Biological Co., California. When the primary culture was started, the medium also contained 0.005% gentamicin (Schering) in order to prevent bacterial contamination. Primary Cell Culture. A small piece of the tumor (approximately 200 mg) was rinsed once in phosphate-buffered isotonic saline, cut free from the encapsulating tissue, and minced as finely as possible with small surgical scissors in a sterilized petri dish. The mashed tumor tissue was dispersed in 16 ml of F-10 medium containing 2.5% fetal calf serum, immediately dispensed into 2 Falcon 3003 petri dishes (100 X 20 mm), and incubated at 370 in a humidified atmosphere of 5% C02/95% air for 2 days. The medium from each dish was saved for hormonal determinations, and the cells which had settled on the petri dish were gently shaken in 1 ml of F-10 medium containing 2.5% fetal calf serum and 0.1% trypsin for 3 min. The cell suspension was centrifuged at low speed and trypsincontaining medium was aspirated. The cell pellet was resuspended in 1 ml of F-10 medium containing 2.5% fetal calf serum. The resuspended cells from each dish were dispersed into two petri dishes: one (a) was cultured in 8 ml of the same medium and the other (b) in 8 ml of F-10 medium containing 20% fetal calf serum. After these replating procedures, the dishes were examined daily for growth and the medium was changed every 3-4 days. Cloning Procedure. During the subsequent 4 weeks of primary culture, it became apparent that in dish 1-b, a homogeneous type of polygonal cells with big nuclei had formed numerous colonies. In this particular dish, no fibroblasts or small epithelioid cells were ever detected. The cell clones showed no
1068
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
Transplantation in Hamster Cheek Pouch. Fang-8 cells (1 X 106 cells per dish) of passage 12 were harvested in 0.2-0.3 ml of medium and inoculated into the cheek pouch of each of five
FIG. 1. (A) Pathology (section) of the primary adrenal adenocarcinoma with hematoma obtained from the patient on May 31, 1973. X110. (B) Another section of the metastatic adrenal carcinoma obtained from the patient on February 17, 1975. X110. (C) Photomicrograph of Fang-8 cloned strain of cells of the first passage. Live culture, unstained, phase contrast. X330. (D) Section of the nodule in hamster cheek pouch 3 weeks after inoculation of Fang-8 clonal cells. Hematoxylin and eosin stain. X110.
cell-contact inhibition. The estrogen content in the medium of dish 1-b (see below) was higher than in the other three dishes, implying that the cells were the most representative of the original tumor cells. One solitary clone containing at least 400 cells was located somewhat distant from the other cells in the dish. After the medium was removed, this interesting clone was easily transferred into a fresh petri dish without disturbing the other cells with the aid of a drop of F-10 medium containing 2.5% fetal calf serum and 0.1% trypsin. During the subsequent 6 weeks of continuous culture, the cloned cells (Fig. 1C) continued to multiply. At this point some medium was saved for estrogen determination and the cells were considered a clonal cell line, designated as the 1st passage of Fang-8 cells. A more strict cloning procedure, to start culture from an isolated single cell origin using stainless steel cylinders (10) was carried out in the subsequent passages, including one after hamster transfer. These cell lines are the same as the first clonal cell line in all aspects.
Serial Subculture. Cloned cells (4 X 105) were transferred into 10 dishes and cultured in F-10 medium supplemented with 2.5% fetal calf serum. On May 21, 1975, (1 X 106 Fang-8 cells) in passage 2 were treated with GIB medium containing 10% calf serum, 10% fetal calf serum, and 10% dimethyl sulfoxide, frozen, and stored in liquid nitrogen in accordance with a procedure described by Shannon and Macy (11). The rest of the cells were used for karyotypic analysis, estrogen and protein determinations, and continued propagation. While beginning cell passage 3, it was discovered that Fang-8 cells showed improved growth in Matalon-modified Eagle's medium containing 10% each of calf and fetal calf sera. For replating, 1 to 2 X 105 cells were transferred to each new dish and cultured in 7-10 ml of medium. The medium was changed twice each week unless otherwise specified. At the time of the submission of this report, the cells have been subcultured to passage 42. During this period of continuous propagation, cells of different passage were studied for karyotypic analysis and also stored in liquid nitrogen. After thawing, the stored cells could be propagated in culturewithout noticeable change in estrogen production and growth.
young male hamsters conditioned to transplantation 24 hr earlier by whole body irradiation (500 rad). Nodules were detected in all five animals after 2 weeks of transplantation and were removed for histologic examination (Fig. ID) 1 week later. A clonal subline (Fang-32) has been propagated ever since and has exhibited no differences from the Fang-8 cells. Growth Curve. Fang-8 cells of the passage 3 and 7 were studied to estimate the population doubling time and the rate of estrogen production. The initial cell number was approximately 5 X 104 cells per dish for a total of 42 dishes; cells were grown in 8 ml of medium as previously described for serial subculture. During the experiment, three dishes were taken every day for 24-hr continued culture in fresh medium. The culture medium was saved for determination of estrogen levels. The cells were washed twice with 3 ml of phosphate-buffered saline, and the washed cells were harvested in exactly 1 ml of phQsphate-buffered saline. An aliquot of 50 Al of the cell suspension was properly diluted in trypsin-containing medium for cell counting in a hemocytometer. The remainder of the cell suspension was sonicated, frozen at -20°, and stored prior to assay for estrogen and cellular protein and DNA content. Protein was determined by the method described by Lowry et al. (12) and DNA by diphenylamine reaction after perchloric acid extraction as described by Burton (13). Estrogen Radioimmunoassay. Estrogen contents of patient serum, culture medium, and cell samples were measured by the radioimmunoassay technique as described by Wu et al. (14), by using a specific antiserum kindly provided by G. E. Abraham of Harbor General Hospital, Torrance, Calif. When medium samples were assayed, the estrogen content of calf and fetal calf sera in control medium before use was determined and subtracted from the estrogen values of the culture media in every experiment. Generally samples were assayed for estrogen without prior fractionation; it was assumed that the total immunoreactive estrogens were measured, although all results were expressed in pg of estradiol-17f3. The sensitivity of assay was 2 pg of estradiol-170; interassay and intra-assay variations were less than 25% and 10%, respectively. Isolation of the three estrogen compounds by LH-20 column chromatography and measurement of each estrogen by specific antiserum was carried out in later experiments. Histological Examination. All the extirpated tumors from patient and hamsters, as well as cloned cells in petri dishes were fixed in isotonic-buffered Formalin (10%, vol/vol), stained with hematoxylin and eosin, and examined under an ordinary light microscope. Tumor slices were also examined for glycogen, lipid, and mucin after proper fixation and staining. Cells in petri dishes were also photographed directly without stain by using a phase contrast condenser. Cultured cells were examined with electron microscope after being fixed- with 5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 postfixed in 2% OS04 in 0.1 M collidine buffer at pH 7:4, and stained with 1% uranyl acetate in 0.1 M sodium maleate buffer at pH 6.0. Karyology. Cells were cultured for 48 hr after transfer to new dishes and treated with Colcemid (GIBCO) (0.2,ug/ml) for 1-4 hr at 370, except in three experiments in which no Colcemid was used. Cells undergoing mitosis were harvested in hypotonic KCI solution (0.85%) and packed by low-speed centrifugation. The cell pellets were fixed for 20 min in methanol-acetic acid (3:1, vol/vol). After three changes of fixative the cells were dropped onto wet slides, dried on a hot-plate (60°), stained with Giemsa or quinacrine, and then examined under light micro-
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
1069
Table 1. Estrogen production by primary culture and Fang-8 clonal cell line
-,Z.
t C
7~I '11
Date
Culture and passage
Protein (jg/dish)
24-Hour estrogen production (pg/dish)
Primary ~ ~ ~
~~~~~~~~~~-
~
~
~
~
~
2-19-75 3-12-75
FIG. 2. (A) Photomicrograph of the ringed colony of Fang-8 clonal cells in culture. Note the multilayer proliferation and ringed configuration. Live culture, unstained, phase contrast. (B) Photomicrograph of Fang-8 clonal cells from the same culture after fixing and staining. Note the formation of a ductal luminal membrane connecting the neighboring cells. X120.
scope for karyotype analysis. Effort was also made to identify the presence of the Y chromosome by the fluorescence method of Pearson et al. (15). RESULTS Morphology of a Clonal Strain, Fang-8, of Human Adrenal Cortex Carcinoma Cells. The appearance of Fang-8 cells is shown in Fig. 1C. The cells are polyhedral and spherical in shape. The cell walls are generally rough, with some extending appendages, so that some isolated cells or cells at the edge of a colony may appear in the shape of a trapezoid. Each cell has a prominent, big nucleus which gives the impression that the cell scantily contains cytoplasm surrounding the nucleus. The nucleus contains one or two nucleoli. Characteristically, Fang-8 cells do not form a complete monolayer, as do fibroblasts. Each clone tends to grow and multiply in a three-dimensional configuration; consequently, it is difficult to focus and photograph the cells under ordinary phase-contrast light microscope. There was no cell-figure resemblance between the cell colonies and the original human tumors and tumors from hamster cheek pouch (Fig. 1A, B, and D). These morphological differences result from different environmental (in Vvo and in vitro) conditions. Fang-8 cells have the interesting property of ring-formation. After passage 12, this property became more obvious (Fig. 2A). The cell colonies, after being fixed on petri dishes and stained with hematoxylin and eosin, clearly showed a characteristic ductal luminal membrane connecting the neighboring cells (Fig. 2B). The presence of glycogen was confirmed by periodic acidSchiff reaction with and without diastase pretreatment. Lipid droplets in cells were observed by oil Red-O stain.
t~~~~~~~~n~~g FIG. 3. Electron micrograph showing anastomoses (arrows) between two neighboring cells of the Fang-8 strain. Note also elongated
mitochondria (M) containing tubular cristae, lipid droplets (L), lipofuscin pigment body (lpd), stacks of granular reticulum (sgr), microvilli (mv), and nucleus (N). X3400.
5- 5-75
1 2 la lb 2a 2b Clonal, 1*
500 365 23 65.1 18 21.4
-
-
702 pg/jig of protein
Clonal subculture
5-21-75 10-24-75 1-14-76 1-21-76 2- 4-76 1-14-76 4-19-76 1-21-76 3-10-76 3-11-76 4-19-76 9-10-76 10-29-76 11- 5-76
2 3 5 7 8 10 11 13 14 17 19 36 41
Fang-32,22t
Protein (jig/dish) 375 201 520 90 630 660 720 410 400 730
810 570 950 410
Cell
Medium
0.21 0.17 0.19 0.26 0.09 0.09 0.20 0.15 0.17 0.08 0.11 0.11 0.08 0.22
2.80 3.52 1.60 0.89 3.37 2.61 2.15 4.90 4.00 1.58 1.55 2.89 1.47 4.58
Data represent total estrogen minus control values, which were less than 150 pg/dish. * Clonal strain of the cell line was derived from the primary culture in petri dish 1-b. t Subline after transfer from hamster cheek pouch.
Electron micrographs of Fang-8 cells show the features of a differentiated adrenal cell of zona reticularis as described by Lentz (16). Important features include lipid droplets, often elongated mitochondria containing tubular cristae, stacks of granular reticulum, many lipofuscin pigment bodies, and the presence of glycogen. Of even more interest are the anastomoses which developed between the walls of neighboring cells in culture (Fig. 3). The cell wall has abundant microvilli wherever cellular contents are extruded. There were no viral particles to be seen in any of the cells examined. Estrogen Production in Tumors and Clonal Fang-8 Cell Culture. The original tumor had precipitated the serious feminizing symptoms of the patient and had resulted in constant elevation of his serum estrogen levels. The in vitro production of estrogen by the primary culture and serial subcultures are listed in Table 1. For the primary culture, I was merely interested in confirming the function of estrogen secretion by the cells. After the clonal strain of cells was established, I was able to quantitate the amount of hormone based on cellular protein. Because the antiserum used in the estrogen radioimmunoassay is fairly monospecific to estradiol-170, all samples were assayed for estrogen without prior fractionation. On several occasions, samples were fractionated on Sephadex LH-20 columns before estradiol-170- was measured. The results indicated that 70-90% of the immunoreactive estrogen was indeed estradiol-17fl. The contents of estrone and estriol were 10-25% and 0-5%, respectively. As shown by the growth curve
Cell Biology: Fang
1070
Proc. Natl. Acad. Sci. USA 74 (1977)
results, the population doubling time is estimated as approximately 24 hr before, and during, exponential growth. Karyotypic Analysis. Results of karyotypic analyses of Fang-8 clonal cells as listed in Table 2 indicate that treatment of cells with Colcemid for 4 hr or less did not seem to affect the chromosome frequency distribution, and that serial subcultures were unlikely to alter the karyology. The cells are 100% aneuploid with an unstable karyotype. All cells contain a long, unpaired submetacentric marker chromosome. The other marker chromosomes are currently under investigation. Preliminary examination for the Y chromosome as interphase fluorescence body failed in all cells attempted. However, banding by Giemsa or quinacrine methods clearly indicated the presence of the Y chromosome in some cells. Therefore, further work is required in order to ascertain the seemingly elusive Y chromosome in this clonal cell line which originated from a tumor in a male patient.
soo00
2000 ----
2001 1.
*
C
I c 50- c 500 A
i
a
3 201
200
a 10-
50
5-
0,51 41
c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days in Culture
FIG. 4.
Growth curves of Fang-8 clonal cells of passage 7 in terms
of cell number, cell protein, and DNA content. The lower panel shows
ratios of estrogen production (pg) in 24 hr to gg of cellular protein. Day zero was the day subculture began. Each point represents the average mean value of three dishes.
Of the cultured cells (see below), the amount of estrogen pro-
duced by the cells is strongly dependent upon cell density and age. Nevertheless, the data in Table 1 clearly demonstrate that
the Fang-8 cell line maintained the biochemical function of estrogen production throughout all passages examined. No cortisol, aldosterone, or testosterone was detected from cell cultures by the competitive radioligand assays.
Hamsters
bearing tumors from inoculated Fang-8 clonal cells had mean
serum estrogen levels of 93
+
16.8 (SD) pg/ml, which are sig-
nificantly elevated (P < 0.01) in comparison to normal values of 40
+
6.7 pg/ml (n = 6).
Growth Curve. Fig. 4 shows the growth of Fang-8 clonal cells in terms of cell number, cell protein and DNA content, as well as the relationship of cell growth to estrogen production. A
parallel relationship between cell protein and DNA content as parameters of growth exists during the stage of exponential growth, but cell division always outstrips the syntheses of protein and DNA. For example, on day 9 the cell number has increased to 46 times that of day 1; protein and DNA content have
increased only 6.1 and 4.7 times that of day 1, respectively. After the cell number has reached 6 X
106
per dish, many cells
the number of viable cells declines. The cellular DNA and protein content reached a plateau 1 day later. The ratio of 24-hr estrogen production to cellular protein increased to its highest point after beginning the exponential phase of cell growth. The estrogen to protein ratio was lowest start floating and
and
at the very beginning, also at the very end of the growth curve, and values of the two ratios were very similar, al-
the
though end
the
of the
grow
protein content was at least
30 times higher at the
growth curve than at the beginning. The cells never the dishes as fibroblasts do. Based on these
to confluence in
DISCUSSION The present communication describes a newly established strain of epithelioid cells derived from a metastatic adrenal adenocarcinoma of a male patient. The clonal cell line, designated Fang-8, has been propagated continuously by serial subculture during the past 20 months. The morphology of the cells reveals the characteristic structure of glandular epithelial cells; they do not grow into confluent monolayers, but tend to multiply in a three-dimensional orientation and form colonies of ringed or ductule configuration. Cells often anastomose with one another in culture. Electron microscope pictures of the cells show lipofuscin pigment bodies and elongated mitochondria containing tubular cristae. These results indicate that the parenchymal cells in the zona reticularis of the human adrenal cortex are probably the origin of this cell line. In several subcultures, Fang-8 clonal cells produced at least 1 pg of estrogen per gg of cellular protein in 24 hr but no other steroid hormones which are generally synthesized in the normal adrenal cortex. Because the tumor caused serious feminization in the patient, estrogen production represents phenotypic expression of an inherited genetic marker of Fang-8 clonal cells. Furthermore, hamsters bearing a tumor induced by inoculation of Fang-8 cells had elevated serum levels of estrogen. These results further substantiate the unique biochemical feature of the cell line. Baird et al. (17) have shown that estrone and estradiol-17f3 are secreted by normal human adrenal. However, it is not known whether the zona fasciculata and/or the zona reticularis are responsible for this capability. The estrogensynthesizing property of Fang-8 clonal cells provides circumstantial evidence which implies that the zona reticularis is capable of secreting estrogen. One very worrisome problem which prevails in laboratories where investigators attempt to establish human cell lines is the possible contamination of their strains by HeLa cells. Gartler (18), as a result of studies with electrophoretic variant forms of glucose-6-phosphate dehydrogenase and phosphoglucomutase phenotypes, first suggested that many of the permanent human cell lines may be HeLa cell derivatives. Nelson-Rees et al. (19) applied their criteria of marker chromosomes, lack of the Y chromosome, and glucose-6-phosphate dehydrogenase type A mobility as HeLa characteristics to analyze many well-known cell lines, and concluded that they are actually of HeLa origin.
The Fang-8 cell line was developed in a laboratory which has never worked with HeLa cells, and by this investigator, who is well aware of the danger of such contamination. Enzymatic analyses of Fang-8 clonal cells revealed type A mobility of
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
1071
Table 2. Karyology of Fang-8 cell line Colcemid treatment Passage (hr)
4
4
5
3
7
1
8
0*
10
3
13
1
41
0*
Fang-32, 22t
*
Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes
Chromosome frequency distribution 1 35 1 46 4 26 1 30 1 36 2 39 1 33
1 43 1 50 1 33 1 32 -1 47 1 40 1 37
1 44 2 52 1 42 1 43 2 48 2 42 1 38
1 49 1 53 1 44 2 44 3 50 1 43 1 39
1 50 4 54 2 46 2 46 1 51 2 45 1 44
1 53 1 55 2 48 1 47 5
52 1 46 1 47
2 54 1 57 2 49 1 48 1 53 1 47 1 48
1 55 3 58 3 55 2 49 1 54 2 48 1 50
1 56 1 59 5 56 4 51 1 55 1 51 2 51
8 57 4 60 3 57 1 52 4 56 3 52 1 52
4 58 8 61 2 58 2 53 2 57 4 53 1 54
1 59 11 62 1 59 1 54 2 58 1 54 1 55
11 60 9 63 2 60 3 55 4 60 1 55 1 56
3 61 8 64 2 62 2 56 2 61 4 56 3 57
8 62 4 65 2 63 3 57 2 62 2 58 2 58
8 63 1 66 1 64 1 58 5 63 5 60 1 59
6 2 1 1 64 65 66 67 1 65 1 60 1 64 1 61 4 60
3 66 3 61 1 65 1 62 3 61
1 77 1 62 1 66 1 63 1 62
3 1 2 1 63 64 65 66 1 68 3 1 65 70 4 3 2 1 2 63 64 65 66 67
1 1 1 1 1 3 1 3 3 4 6 5 3 3 1 2 1 CClls Chromosomes 44 51 52 54 56 57 58 59 60 61 62 63 64 65 67 68 75 Cells 1 1 2 2 2 2 2 1 1 1 2 1 2 3 2 2 5 4 3 1 Chromosomes 35 38 43 45 48 50 52 53 55 56 57 58 59 60 61 62 63 64 65 66
* The cell culture was not treated with colcemid. t Subline after transfer from hamster cheek pouch.
glucose-6-phosphate dehydrogenase. This is congruent with the fact that the donor patient was a black. Karyotype analyses of the cells give very different chromosome frequency distribution results from those reported for HeLa (CCL 2), HeLa 229 (CCL 2.1), and HeLa S3 (CCL 2.2) (1). However, identification of the Y chromosome by conventional methods appeared to be uncertain. It is inconceivable that the Fang-8 cell line has been established with the aid of HeLa contaminants or that it should be considered a de facto strain of HeLa (20), unless the patient's metastatic tumor was related to an oncogenic factor of HeLa cell origin. Furthermore, the possibility exists that permanent human cell lines, including HeLa, share certain common karyotypic and enzymatic features but do not actually contaminate one another. Despite this potential problem, the-cell line reported here possesses enough unique biochemical and morphological properties to prove very useful for cancer research and hormonal and related studies in the future. I would like to express my gratitude to Mrs. Joan Chen and Mrs. Ofelia Gomez for their excellent technical assistance. I am grateful to Drs. Edwin L. Kaplan for the surgical tissue, Reuben Matalon for advice on cell culture, W. T. Liang and Elizabeth Dorus for Y chromosome studies, and Robert M. Chen for help in histological studies; and to Ms. Sandra Schmidt for secretarial assistance. Electron microscopic studies were supported by U.S. Public Health Service Grant HD07110. 1. Shannon, J. E. & Macy, M. M. (1975) in Catalogue of Strains: II, Animal Cell Lines (American Type Culture Collection, Rock-
ville, Md). 2. Buonassisi, V., Sato, G. & Cohen, A. I. (1962) Proc. Natl. Acad. Sci. USA 48, 1184-1190.
3. Yasumura, Y., Tashjian, A. H., Jr. & Sato, G. H. (1966) Science 154, 1186-1189. 4. Leibovitz, A., McCombs, W. B., III, Johnston, D., McCoy, C. E. & Stinson, J. C. (1973) J. Natl. Cancer Inst. 51, 691-697. 5. Patillo, R. A. & Gey, G. 0. (1968) Cancer Res. 28, 1231-1236. 6. Patillo, R. A., Gey, G. O., Delfs, E., Huang, W. Y., Hause, L., Gerancis, J., Knoth, M., Amatruda, J., Bertino, J., Friesen, H. G. & Mattingly, R. F. (1971) Ann. N.Y. Acad. Sci. 172,288-290. 7. Gabrilove, J. L., Sharma, D. C., Wotiz, H. H. & Dorfman, R. I. (1965) Medicine 44,37-79. 8. Ham, R. G. (1963) Exp. Cell Res. 29,515-526. 9. Matalon, R. & Dorfman, A. (1966) Proc. Natl. Acad. Sci. USA 56, 1310-1316. 10. Tashjian, A. H., Jr., Yasumura, Y., Levine, L., Sato, G. H. &
Parker, M. L. (1968) Endocrinology 82, 342-352. 11. Shannon, J. E. & Macy, M. M. (1973) in Tissue Culture: Methods and Applications, eds. Kruse, P. F. & Patterson, M. K. (Academic
Press, New York), pp. 712-718. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193, 265-275.
13. Burton, K. (1956) Biochem. J. 62,315-323. 14. Wu, C. H., Lundy, L. E. & Lee, S. G. (1973) Am. J. Obstet. Gynecol. 115, 169-180. 15. Pearson, P. L., Bobrow, M. & Vosa, C. G. (1970) Nature 226, 78-80. 16. Lentz, T. L. (1971) in Cell Fine Structure (W. B. Saunders Co., Philadelphia, Pa.), pp. 332-339. 17. Baird, D. T., Uno, A. & Melby, J. C. (1969) J. Endocrinol. 45, 135-136. 18. Gartler, S. M. (1968) Nature 217,750-751. 19. Nelson-Rees, W. A., Flandermeyer, R. R. & Hawthorne, P. K. (1974) Science 184, 1093-1096. 20. Nelson-Rees, W. A. & Flandermeyer, R. R. (1976) Science 191, 96-98.
Establishment and characterization of a strain of human adrenal tumor cells that secrete estrogen (Fang-8 clonal cell line/epithelioid cells)
VICTOR S. FANG Department of Medicine, The University of Chicago, Chicago, Illinois 60637
Communicated by Elwood V. Jensen, December 3, 1976
We report the establishment of a new cell line, ABSTRACT designated Fang-8, which originated from a human adrenal adenocarcinoma. It has been continuously propagated during the past 20 months. The cells exhibit an epithelioid morphology, resembling the cell structure of the zona reticularis when viewed by electron microscopy. Since passage 12, the cells showed a characteristic ring-forming property in culture. The cells produce neither testosterone, mineralocorticosteroids, nor glucocorticosteroids; instead, they possess the unique function of estrogen production. Karyotypic analysis revealed a 100% aneuploid, unstable karyotype in the hyperdiploid stem line. Identification of the Y chromosome was uncertain. In view of these characteristics, Fang-8 represents a new human cell line, unlikely to be contaminated with HeLa strains, and different from another cell line derived from human adrenal cortex adenocarcinoma, namely SW-13 (ATCC CCL 105).
Among the many mammalian cell lines deposited with the American Type Culture Collection (ATCC) (1), very few have maintained some unique biochemical function which can be utilized for further characterization of the cells in an in vitro system. Cell lines of endocrine tissue origin, by retaining hormonal production, provide an ideal model in this respect. However, due to either a phenotypic change or inadequate environmental conditions, the tissue-specific function could easily disappear during serial propagation of the cultured animal cells. In order to overcome these disadvantages, Buonassisi et al. (2) developed a method of alternate culture and animal passage, and consequently, several functioning rodent endocrine cell lines were successfully established (3). Obviously, such a technique cannot be used to establish human cell lines. Of the 53 human cell lines which have so far been deposited with ATCC (1), only one line, SW-13 (CCL 105) was derived from human adrenal cortex adenocarcinoma (4); its hormonal production was not studied. Another interesting cell line, BeWo (CCL 98), derived from choriocarcinoma (5), maintained hormone-synthesizing functions, and produced human chorionic gonadotropin and estrogens (6). During the past 2 years, I attempted to develop stable human cell lines from various neoplastic tissues which were known to produce hormones. In the present communication, I report one such clonal strain of human cells that has been propagated by serial subcultures in my laboratory and continuously produces estrogen. MATERIALS AND METHODS The cells were obtained from a patient with the following history: A.W., Jr., a black male, father of three children, noticed diminished libido, impotency, and mammary gland enlargement since 1972. He did not seek further medical treatment until May 31, 1973 when he came to the emergency room of Billings Hospital, and complained of acute abdominal pain. By exploratory laparotomy, a ruptured tumor of the right adrenal gland with large retroperitoneal hemorrhage and abscess was discovered. Histologic examination revealed an adrenal ade1067
nocarcinoma with hematoma (Fig. 1A). His postoperative serum levels of estrogen as measured by radioimmunoassay technique ranged from 90 to 240 pg/ml (normal male value in our laboratory is 50 + 15 pg/ml, mean L SD). Values for the other adrenal hormones were within normal limits. Beginning in August, 1973, the patient was treated with 5 g of mitotane, daily, but his serum estrogen levels were progressively elevated. In January, 1974 he developed hypertension. In August, 1974 hepatic metastases were detected. On February 17, 1975, a palliative gastrojejunostomy was performed to relieve gastric outlet obstruction by tumor compression. A tumor mass (4 X 4 cm) at right side was removed for histologic examination which revealed adrenal carcinoma (Fig. 1B). A small piece of the same tumor mass was cultured. The patient died of renal failure on April 15, 1975, at age 30. He suffered a metastatic feminizing adrenocortical adenocarcinoma, a relatively rare disease with poor prognosis (for a review see ref. 7). Culture Media. The medium used for the primary culture was F-10 synthetic culture medium (8) supplemented with 20% or 2.5% fetal calf serum. The medium used for serial subcultures was Matalon modified Eagle's medium (9), supplemented with 10% each of calf and fetal calf sera. All media were purchased from Grand Island Biological Co., California. When the primary culture was started, the medium also contained 0.005% gentamicin (Schering) in order to prevent bacterial contamination. Primary Cell Culture. A small piece of the tumor (approximately 200 mg) was rinsed once in phosphate-buffered isotonic saline, cut free from the encapsulating tissue, and minced as finely as possible with small surgical scissors in a sterilized petri dish. The mashed tumor tissue was dispersed in 16 ml of F-10 medium containing 2.5% fetal calf serum, immediately dispensed into 2 Falcon 3003 petri dishes (100 X 20 mm), and incubated at 370 in a humidified atmosphere of 5% C02/95% air for 2 days. The medium from each dish was saved for hormonal determinations, and the cells which had settled on the petri dish were gently shaken in 1 ml of F-10 medium containing 2.5% fetal calf serum and 0.1% trypsin for 3 min. The cell suspension was centrifuged at low speed and trypsincontaining medium was aspirated. The cell pellet was resuspended in 1 ml of F-10 medium containing 2.5% fetal calf serum. The resuspended cells from each dish were dispersed into two petri dishes: one (a) was cultured in 8 ml of the same medium and the other (b) in 8 ml of F-10 medium containing 20% fetal calf serum. After these replating procedures, the dishes were examined daily for growth and the medium was changed every 3-4 days. Cloning Procedure. During the subsequent 4 weeks of primary culture, it became apparent that in dish 1-b, a homogeneous type of polygonal cells with big nuclei had formed numerous colonies. In this particular dish, no fibroblasts or small epithelioid cells were ever detected. The cell clones showed no
1068
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
Transplantation in Hamster Cheek Pouch. Fang-8 cells (1 X 106 cells per dish) of passage 12 were harvested in 0.2-0.3 ml of medium and inoculated into the cheek pouch of each of five
FIG. 1. (A) Pathology (section) of the primary adrenal adenocarcinoma with hematoma obtained from the patient on May 31, 1973. X110. (B) Another section of the metastatic adrenal carcinoma obtained from the patient on February 17, 1975. X110. (C) Photomicrograph of Fang-8 cloned strain of cells of the first passage. Live culture, unstained, phase contrast. X330. (D) Section of the nodule in hamster cheek pouch 3 weeks after inoculation of Fang-8 clonal cells. Hematoxylin and eosin stain. X110.
cell-contact inhibition. The estrogen content in the medium of dish 1-b (see below) was higher than in the other three dishes, implying that the cells were the most representative of the original tumor cells. One solitary clone containing at least 400 cells was located somewhat distant from the other cells in the dish. After the medium was removed, this interesting clone was easily transferred into a fresh petri dish without disturbing the other cells with the aid of a drop of F-10 medium containing 2.5% fetal calf serum and 0.1% trypsin. During the subsequent 6 weeks of continuous culture, the cloned cells (Fig. 1C) continued to multiply. At this point some medium was saved for estrogen determination and the cells were considered a clonal cell line, designated as the 1st passage of Fang-8 cells. A more strict cloning procedure, to start culture from an isolated single cell origin using stainless steel cylinders (10) was carried out in the subsequent passages, including one after hamster transfer. These cell lines are the same as the first clonal cell line in all aspects.
Serial Subculture. Cloned cells (4 X 105) were transferred into 10 dishes and cultured in F-10 medium supplemented with 2.5% fetal calf serum. On May 21, 1975, (1 X 106 Fang-8 cells) in passage 2 were treated with GIB medium containing 10% calf serum, 10% fetal calf serum, and 10% dimethyl sulfoxide, frozen, and stored in liquid nitrogen in accordance with a procedure described by Shannon and Macy (11). The rest of the cells were used for karyotypic analysis, estrogen and protein determinations, and continued propagation. While beginning cell passage 3, it was discovered that Fang-8 cells showed improved growth in Matalon-modified Eagle's medium containing 10% each of calf and fetal calf sera. For replating, 1 to 2 X 105 cells were transferred to each new dish and cultured in 7-10 ml of medium. The medium was changed twice each week unless otherwise specified. At the time of the submission of this report, the cells have been subcultured to passage 42. During this period of continuous propagation, cells of different passage were studied for karyotypic analysis and also stored in liquid nitrogen. After thawing, the stored cells could be propagated in culturewithout noticeable change in estrogen production and growth.
young male hamsters conditioned to transplantation 24 hr earlier by whole body irradiation (500 rad). Nodules were detected in all five animals after 2 weeks of transplantation and were removed for histologic examination (Fig. ID) 1 week later. A clonal subline (Fang-32) has been propagated ever since and has exhibited no differences from the Fang-8 cells. Growth Curve. Fang-8 cells of the passage 3 and 7 were studied to estimate the population doubling time and the rate of estrogen production. The initial cell number was approximately 5 X 104 cells per dish for a total of 42 dishes; cells were grown in 8 ml of medium as previously described for serial subculture. During the experiment, three dishes were taken every day for 24-hr continued culture in fresh medium. The culture medium was saved for determination of estrogen levels. The cells were washed twice with 3 ml of phosphate-buffered saline, and the washed cells were harvested in exactly 1 ml of phQsphate-buffered saline. An aliquot of 50 Al of the cell suspension was properly diluted in trypsin-containing medium for cell counting in a hemocytometer. The remainder of the cell suspension was sonicated, frozen at -20°, and stored prior to assay for estrogen and cellular protein and DNA content. Protein was determined by the method described by Lowry et al. (12) and DNA by diphenylamine reaction after perchloric acid extraction as described by Burton (13). Estrogen Radioimmunoassay. Estrogen contents of patient serum, culture medium, and cell samples were measured by the radioimmunoassay technique as described by Wu et al. (14), by using a specific antiserum kindly provided by G. E. Abraham of Harbor General Hospital, Torrance, Calif. When medium samples were assayed, the estrogen content of calf and fetal calf sera in control medium before use was determined and subtracted from the estrogen values of the culture media in every experiment. Generally samples were assayed for estrogen without prior fractionation; it was assumed that the total immunoreactive estrogens were measured, although all results were expressed in pg of estradiol-17f3. The sensitivity of assay was 2 pg of estradiol-170; interassay and intra-assay variations were less than 25% and 10%, respectively. Isolation of the three estrogen compounds by LH-20 column chromatography and measurement of each estrogen by specific antiserum was carried out in later experiments. Histological Examination. All the extirpated tumors from patient and hamsters, as well as cloned cells in petri dishes were fixed in isotonic-buffered Formalin (10%, vol/vol), stained with hematoxylin and eosin, and examined under an ordinary light microscope. Tumor slices were also examined for glycogen, lipid, and mucin after proper fixation and staining. Cells in petri dishes were also photographed directly without stain by using a phase contrast condenser. Cultured cells were examined with electron microscope after being fixed- with 5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 postfixed in 2% OS04 in 0.1 M collidine buffer at pH 7:4, and stained with 1% uranyl acetate in 0.1 M sodium maleate buffer at pH 6.0. Karyology. Cells were cultured for 48 hr after transfer to new dishes and treated with Colcemid (GIBCO) (0.2,ug/ml) for 1-4 hr at 370, except in three experiments in which no Colcemid was used. Cells undergoing mitosis were harvested in hypotonic KCI solution (0.85%) and packed by low-speed centrifugation. The cell pellets were fixed for 20 min in methanol-acetic acid (3:1, vol/vol). After three changes of fixative the cells were dropped onto wet slides, dried on a hot-plate (60°), stained with Giemsa or quinacrine, and then examined under light micro-
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
1069
Table 1. Estrogen production by primary culture and Fang-8 clonal cell line
-,Z.
t C
7~I '11
Date
Culture and passage
Protein (jg/dish)
24-Hour estrogen production (pg/dish)
Primary ~ ~ ~
~~~~~~~~~~-
~
~
~
~
~
2-19-75 3-12-75
FIG. 2. (A) Photomicrograph of the ringed colony of Fang-8 clonal cells in culture. Note the multilayer proliferation and ringed configuration. Live culture, unstained, phase contrast. (B) Photomicrograph of Fang-8 clonal cells from the same culture after fixing and staining. Note the formation of a ductal luminal membrane connecting the neighboring cells. X120.
scope for karyotype analysis. Effort was also made to identify the presence of the Y chromosome by the fluorescence method of Pearson et al. (15). RESULTS Morphology of a Clonal Strain, Fang-8, of Human Adrenal Cortex Carcinoma Cells. The appearance of Fang-8 cells is shown in Fig. 1C. The cells are polyhedral and spherical in shape. The cell walls are generally rough, with some extending appendages, so that some isolated cells or cells at the edge of a colony may appear in the shape of a trapezoid. Each cell has a prominent, big nucleus which gives the impression that the cell scantily contains cytoplasm surrounding the nucleus. The nucleus contains one or two nucleoli. Characteristically, Fang-8 cells do not form a complete monolayer, as do fibroblasts. Each clone tends to grow and multiply in a three-dimensional configuration; consequently, it is difficult to focus and photograph the cells under ordinary phase-contrast light microscope. There was no cell-figure resemblance between the cell colonies and the original human tumors and tumors from hamster cheek pouch (Fig. 1A, B, and D). These morphological differences result from different environmental (in Vvo and in vitro) conditions. Fang-8 cells have the interesting property of ring-formation. After passage 12, this property became more obvious (Fig. 2A). The cell colonies, after being fixed on petri dishes and stained with hematoxylin and eosin, clearly showed a characteristic ductal luminal membrane connecting the neighboring cells (Fig. 2B). The presence of glycogen was confirmed by periodic acidSchiff reaction with and without diastase pretreatment. Lipid droplets in cells were observed by oil Red-O stain.
t~~~~~~~~n~~g FIG. 3. Electron micrograph showing anastomoses (arrows) between two neighboring cells of the Fang-8 strain. Note also elongated
mitochondria (M) containing tubular cristae, lipid droplets (L), lipofuscin pigment body (lpd), stacks of granular reticulum (sgr), microvilli (mv), and nucleus (N). X3400.
5- 5-75
1 2 la lb 2a 2b Clonal, 1*
500 365 23 65.1 18 21.4
-
-
702 pg/jig of protein
Clonal subculture
5-21-75 10-24-75 1-14-76 1-21-76 2- 4-76 1-14-76 4-19-76 1-21-76 3-10-76 3-11-76 4-19-76 9-10-76 10-29-76 11- 5-76
2 3 5 7 8 10 11 13 14 17 19 36 41
Fang-32,22t
Protein (jig/dish) 375 201 520 90 630 660 720 410 400 730
810 570 950 410
Cell
Medium
0.21 0.17 0.19 0.26 0.09 0.09 0.20 0.15 0.17 0.08 0.11 0.11 0.08 0.22
2.80 3.52 1.60 0.89 3.37 2.61 2.15 4.90 4.00 1.58 1.55 2.89 1.47 4.58
Data represent total estrogen minus control values, which were less than 150 pg/dish. * Clonal strain of the cell line was derived from the primary culture in petri dish 1-b. t Subline after transfer from hamster cheek pouch.
Electron micrographs of Fang-8 cells show the features of a differentiated adrenal cell of zona reticularis as described by Lentz (16). Important features include lipid droplets, often elongated mitochondria containing tubular cristae, stacks of granular reticulum, many lipofuscin pigment bodies, and the presence of glycogen. Of even more interest are the anastomoses which developed between the walls of neighboring cells in culture (Fig. 3). The cell wall has abundant microvilli wherever cellular contents are extruded. There were no viral particles to be seen in any of the cells examined. Estrogen Production in Tumors and Clonal Fang-8 Cell Culture. The original tumor had precipitated the serious feminizing symptoms of the patient and had resulted in constant elevation of his serum estrogen levels. The in vitro production of estrogen by the primary culture and serial subcultures are listed in Table 1. For the primary culture, I was merely interested in confirming the function of estrogen secretion by the cells. After the clonal strain of cells was established, I was able to quantitate the amount of hormone based on cellular protein. Because the antiserum used in the estrogen radioimmunoassay is fairly monospecific to estradiol-170, all samples were assayed for estrogen without prior fractionation. On several occasions, samples were fractionated on Sephadex LH-20 columns before estradiol-170- was measured. The results indicated that 70-90% of the immunoreactive estrogen was indeed estradiol-17fl. The contents of estrone and estriol were 10-25% and 0-5%, respectively. As shown by the growth curve
Cell Biology: Fang
1070
Proc. Natl. Acad. Sci. USA 74 (1977)
results, the population doubling time is estimated as approximately 24 hr before, and during, exponential growth. Karyotypic Analysis. Results of karyotypic analyses of Fang-8 clonal cells as listed in Table 2 indicate that treatment of cells with Colcemid for 4 hr or less did not seem to affect the chromosome frequency distribution, and that serial subcultures were unlikely to alter the karyology. The cells are 100% aneuploid with an unstable karyotype. All cells contain a long, unpaired submetacentric marker chromosome. The other marker chromosomes are currently under investigation. Preliminary examination for the Y chromosome as interphase fluorescence body failed in all cells attempted. However, banding by Giemsa or quinacrine methods clearly indicated the presence of the Y chromosome in some cells. Therefore, further work is required in order to ascertain the seemingly elusive Y chromosome in this clonal cell line which originated from a tumor in a male patient.
soo00
2000 ----
2001 1.
*
C
I c 50- c 500 A
i
a
3 201
200
a 10-
50
5-
0,51 41
c
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Days in Culture
FIG. 4.
Growth curves of Fang-8 clonal cells of passage 7 in terms
of cell number, cell protein, and DNA content. The lower panel shows
ratios of estrogen production (pg) in 24 hr to gg of cellular protein. Day zero was the day subculture began. Each point represents the average mean value of three dishes.
Of the cultured cells (see below), the amount of estrogen pro-
duced by the cells is strongly dependent upon cell density and age. Nevertheless, the data in Table 1 clearly demonstrate that
the Fang-8 cell line maintained the biochemical function of estrogen production throughout all passages examined. No cortisol, aldosterone, or testosterone was detected from cell cultures by the competitive radioligand assays.
Hamsters
bearing tumors from inoculated Fang-8 clonal cells had mean
serum estrogen levels of 93
+
16.8 (SD) pg/ml, which are sig-
nificantly elevated (P < 0.01) in comparison to normal values of 40
+
6.7 pg/ml (n = 6).
Growth Curve. Fig. 4 shows the growth of Fang-8 clonal cells in terms of cell number, cell protein and DNA content, as well as the relationship of cell growth to estrogen production. A
parallel relationship between cell protein and DNA content as parameters of growth exists during the stage of exponential growth, but cell division always outstrips the syntheses of protein and DNA. For example, on day 9 the cell number has increased to 46 times that of day 1; protein and DNA content have
increased only 6.1 and 4.7 times that of day 1, respectively. After the cell number has reached 6 X
106
per dish, many cells
the number of viable cells declines. The cellular DNA and protein content reached a plateau 1 day later. The ratio of 24-hr estrogen production to cellular protein increased to its highest point after beginning the exponential phase of cell growth. The estrogen to protein ratio was lowest start floating and
and
at the very beginning, also at the very end of the growth curve, and values of the two ratios were very similar, al-
the
though end
the
of the
grow
protein content was at least
30 times higher at the
growth curve than at the beginning. The cells never the dishes as fibroblasts do. Based on these
to confluence in
DISCUSSION The present communication describes a newly established strain of epithelioid cells derived from a metastatic adrenal adenocarcinoma of a male patient. The clonal cell line, designated Fang-8, has been propagated continuously by serial subculture during the past 20 months. The morphology of the cells reveals the characteristic structure of glandular epithelial cells; they do not grow into confluent monolayers, but tend to multiply in a three-dimensional orientation and form colonies of ringed or ductule configuration. Cells often anastomose with one another in culture. Electron microscope pictures of the cells show lipofuscin pigment bodies and elongated mitochondria containing tubular cristae. These results indicate that the parenchymal cells in the zona reticularis of the human adrenal cortex are probably the origin of this cell line. In several subcultures, Fang-8 clonal cells produced at least 1 pg of estrogen per gg of cellular protein in 24 hr but no other steroid hormones which are generally synthesized in the normal adrenal cortex. Because the tumor caused serious feminization in the patient, estrogen production represents phenotypic expression of an inherited genetic marker of Fang-8 clonal cells. Furthermore, hamsters bearing a tumor induced by inoculation of Fang-8 cells had elevated serum levels of estrogen. These results further substantiate the unique biochemical feature of the cell line. Baird et al. (17) have shown that estrone and estradiol-17f3 are secreted by normal human adrenal. However, it is not known whether the zona fasciculata and/or the zona reticularis are responsible for this capability. The estrogensynthesizing property of Fang-8 clonal cells provides circumstantial evidence which implies that the zona reticularis is capable of secreting estrogen. One very worrisome problem which prevails in laboratories where investigators attempt to establish human cell lines is the possible contamination of their strains by HeLa cells. Gartler (18), as a result of studies with electrophoretic variant forms of glucose-6-phosphate dehydrogenase and phosphoglucomutase phenotypes, first suggested that many of the permanent human cell lines may be HeLa cell derivatives. Nelson-Rees et al. (19) applied their criteria of marker chromosomes, lack of the Y chromosome, and glucose-6-phosphate dehydrogenase type A mobility as HeLa characteristics to analyze many well-known cell lines, and concluded that they are actually of HeLa origin.
The Fang-8 cell line was developed in a laboratory which has never worked with HeLa cells, and by this investigator, who is well aware of the danger of such contamination. Enzymatic analyses of Fang-8 clonal cells revealed type A mobility of
Cell Biology: Fang
Proc. Natl. Acad. Sci. USA 74 (1977)
1071
Table 2. Karyology of Fang-8 cell line Colcemid treatment Passage (hr)
4
4
5
3
7
1
8
0*
10
3
13
1
41
0*
Fang-32, 22t
*
Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes Cells Chromosomes
Chromosome frequency distribution 1 35 1 46 4 26 1 30 1 36 2 39 1 33
1 43 1 50 1 33 1 32 -1 47 1 40 1 37
1 44 2 52 1 42 1 43 2 48 2 42 1 38
1 49 1 53 1 44 2 44 3 50 1 43 1 39
1 50 4 54 2 46 2 46 1 51 2 45 1 44
1 53 1 55 2 48 1 47 5
52 1 46 1 47
2 54 1 57 2 49 1 48 1 53 1 47 1 48
1 55 3 58 3 55 2 49 1 54 2 48 1 50
1 56 1 59 5 56 4 51 1 55 1 51 2 51
8 57 4 60 3 57 1 52 4 56 3 52 1 52
4 58 8 61 2 58 2 53 2 57 4 53 1 54
1 59 11 62 1 59 1 54 2 58 1 54 1 55
11 60 9 63 2 60 3 55 4 60 1 55 1 56
3 61 8 64 2 62 2 56 2 61 4 56 3 57
8 62 4 65 2 63 3 57 2 62 2 58 2 58
8 63 1 66 1 64 1 58 5 63 5 60 1 59
6 2 1 1 64 65 66 67 1 65 1 60 1 64 1 61 4 60
3 66 3 61 1 65 1 62 3 61
1 77 1 62 1 66 1 63 1 62
3 1 2 1 63 64 65 66 1 68 3 1 65 70 4 3 2 1 2 63 64 65 66 67
1 1 1 1 1 3 1 3 3 4 6 5 3 3 1 2 1 CClls Chromosomes 44 51 52 54 56 57 58 59 60 61 62 63 64 65 67 68 75 Cells 1 1 2 2 2 2 2 1 1 1 2 1 2 3 2 2 5 4 3 1 Chromosomes 35 38 43 45 48 50 52 53 55 56 57 58 59 60 61 62 63 64 65 66
* The cell culture was not treated with colcemid. t Subline after transfer from hamster cheek pouch.
glucose-6-phosphate dehydrogenase. This is congruent with the fact that the donor patient was a black. Karyotype analyses of the cells give very different chromosome frequency distribution results from those reported for HeLa (CCL 2), HeLa 229 (CCL 2.1), and HeLa S3 (CCL 2.2) (1). However, identification of the Y chromosome by conventional methods appeared to be uncertain. It is inconceivable that the Fang-8 cell line has been established with the aid of HeLa contaminants or that it should be considered a de facto strain of HeLa (20), unless the patient's metastatic tumor was related to an oncogenic factor of HeLa cell origin. Furthermore, the possibility exists that permanent human cell lines, including HeLa, share certain common karyotypic and enzymatic features but do not actually contaminate one another. Despite this potential problem, the-cell line reported here possesses enough unique biochemical and morphological properties to prove very useful for cancer research and hormonal and related studies in the future. I would like to express my gratitude to Mrs. Joan Chen and Mrs. Ofelia Gomez for their excellent technical assistance. I am grateful to Drs. Edwin L. Kaplan for the surgical tissue, Reuben Matalon for advice on cell culture, W. T. Liang and Elizabeth Dorus for Y chromosome studies, and Robert M. Chen for help in histological studies; and to Ms. Sandra Schmidt for secretarial assistance. Electron microscopic studies were supported by U.S. Public Health Service Grant HD07110. 1. Shannon, J. E. & Macy, M. M. (1975) in Catalogue of Strains: II, Animal Cell Lines (American Type Culture Collection, Rock-
ville, Md). 2. Buonassisi, V., Sato, G. & Cohen, A. I. (1962) Proc. Natl. Acad. Sci. USA 48, 1184-1190.
3. Yasumura, Y., Tashjian, A. H., Jr. & Sato, G. H. (1966) Science 154, 1186-1189. 4. Leibovitz, A., McCombs, W. B., III, Johnston, D., McCoy, C. E. & Stinson, J. C. (1973) J. Natl. Cancer Inst. 51, 691-697. 5. Patillo, R. A. & Gey, G. 0. (1968) Cancer Res. 28, 1231-1236. 6. Patillo, R. A., Gey, G. O., Delfs, E., Huang, W. Y., Hause, L., Gerancis, J., Knoth, M., Amatruda, J., Bertino, J., Friesen, H. G. & Mattingly, R. F. (1971) Ann. N.Y. Acad. Sci. 172,288-290. 7. Gabrilove, J. L., Sharma, D. C., Wotiz, H. H. & Dorfman, R. I. (1965) Medicine 44,37-79. 8. Ham, R. G. (1963) Exp. Cell Res. 29,515-526. 9. Matalon, R. & Dorfman, A. (1966) Proc. Natl. Acad. Sci. USA 56, 1310-1316. 10. Tashjian, A. H., Jr., Yasumura, Y., Levine, L., Sato, G. H. &
Parker, M. L. (1968) Endocrinology 82, 342-352. 11. Shannon, J. E. & Macy, M. M. (1973) in Tissue Culture: Methods and Applications, eds. Kruse, P. F. & Patterson, M. K. (Academic
Press, New York), pp. 712-718. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193, 265-275.
13. Burton, K. (1956) Biochem. J. 62,315-323. 14. Wu, C. H., Lundy, L. E. & Lee, S. G. (1973) Am. J. Obstet. Gynecol. 115, 169-180. 15. Pearson, P. L., Bobrow, M. & Vosa, C. G. (1970) Nature 226, 78-80. 16. Lentz, T. L. (1971) in Cell Fine Structure (W. B. Saunders Co., Philadelphia, Pa.), pp. 332-339. 17. Baird, D. T., Uno, A. & Melby, J. C. (1969) J. Endocrinol. 45, 135-136. 18. Gartler, S. M. (1968) Nature 217,750-751. 19. Nelson-Rees, W. A., Flandermeyer, R. R. & Hawthorne, P. K. (1974) Science 184, 1093-1096. 20. Nelson-Rees, W. A. & Flandermeyer, R. R. (1976) Science 191, 96-98.
