- Email: [email protected]
Establishment and characterization of the cell-line of a human edonmetrial adenoacanthoma
Europ. J. CancerVol. 13, pp. 253-259. Pergamon Press 1977. Printed in Great Britain
Establishment and Characterization of the Cell-Line of a Human Edonmetrial Adenoacanthoma HIROYUKI K U R A M O T O and MIEKO HAMANO Department of Obstetrics and Gynecology, and Preventive Medicine System, School of Medicine, Kitasato University, Sagamihara, Japan Abstract A new cell-line of a human endometrial adenoacanthoma, designated as HEC-6, has been established and propagates well with a permanent stability. The population doubling time of the line is calculated as 52 hr. The in vitro morphology reveals the features of a well-differentiated adenocarcinoma such as a jig-saw puzzlelike arrangement and theformation of an acinous cell-free space in the monolayer culture. Each cell consists of a round atypical nucleus with 2 or 3 marked nudeoli and a vacuolated cytoplasm. A piling-up tendency is a prominent feature and forms an obvious palisade-like or glandular structure. The karyotype of the stem cell is a pseudo-diploid in which variant chromosomes occur at random in various groups. When transplanted into a hamster cheek pouch, line HEC-6 demonstrated an adenocarcinoma in addition to a stratified epithelial component. The original characteristics of an adenoacanthoma of the corpus uteri have been maintained in the culture system.
from a 48-year-old patient with carcinoma of the body of the uterus. This nulliparous patient has been well with regular menstrual cycle until she first complained of intermittent bloody discharge for 2 months duration. She used to live in Hiroshima and was 25 when an atomic bomb exploded there during World War II. An endometrial curettage showed an adenoacanthoma of the endometrium and the portion of a squamous epithelium was rather atypical in shape (Fig. 1). The patient was treated by total hysterectomy, bilateral salpingo-oophorectomy and pelvic lymphadenectomy on J u l y 24, 1970. The uterus, the size of a small goose egg, showed a circumscribed tumour on the fundus, bulging into the cavity. The specimen adjacent to the cultured material revealed a well-differentiated adenocarcinoma.
INTRODUCTION
THE ENDOMETRIUM, a target of sex steroid hormones, undergoes a cyclic alteration by the effects of the hormones. It is important to know if the endometrial gland still preserves the ability as a hormone target even after its maligant transformation. The various characteristics of h u m a n endometrial carcin o m a should include the relation to hormones, however, because of the lack of a suitable experimental tool they have yet to be identified in detail. An in vitro cellular system might contribute to these purposes, supplying a convenient research method. We have established line HEC-1 originating from an adenocarcinoma of the corpus uteri and identified various features of the tumour using this in vitro experimental system [1-3]. The present paper reports the establishment of the new corpus tumour cell-line, its morphology, cytogenetic features and heterotransplantability as well as tissue reproducibility.
Culture method A monolayer culture method was used. The tumour material from the excised uterus was first washed twice in a phosphate buffered solution (Ca 2+ and Mg 2+ free) and minced into fragments with fine scissors and ventilated in 0"25% trypsin solution. The fluid and the minced tumour were stirred slowly by a magnetic stirrer (500rev/min) for 15rain.
MATERIAL A N D M E T H O D S Case history of the culture material The fresh tumour material was obtained
Accepted 11 October 1976. 253
254
Hiroyuki Kuramoto and Mieko Hamano
The trypsin solution was changed 4 times. Then the turbid fluid in which the explant materials were dispersed into a single-cell suspension was aspirated and centrifuged twice after mixing with a culture medium. Subsequently, the sediment was resuspended in medium and placed on a stationary culture in 5% carbon dioxide and 95% air or, occasionally, in a closed vessel. Subcultures were made after treating them with solution of 0.1% trypsin and 0"02% ethylendiamine tetraacetate (EDTA).
lated into plastic Petri dishes ( 6 0 m m in diameter; Lux Plastics) and grown as replicate cultures [4] in a humidified atmosphere containing 5% COz and 95% air. Every 2 days after the subculture, two dishes were harvested to count the number of cells. The media in the remaining dishes was changed. The number of cells in each dish was counted 4 times, using a Burker-Tfirk counter plate. The mean was calculated for each dish and for every harvest. The data was plotted as a semilogarithmic graph.
Growth medium Tissue culture medium No. 199 supplemented with 15% calf serum was used, containing penicillin in 100 U/ml and streptomycin in 100 pg/ml.
Transplantation into a hamster A female hamster, 4 weeks old and weighing about 50 grams, was used for the experiment. The cell cultures were dispersed into a suspension of single cells by the trypsin-EDTA solution, and then were washed with culture media and centrifuged twice. The cells, 42.0 or 75-0x 105 in number, were injected into the submucosa of a hamster cheek pouch by a syringe with a No. 22 gauge needle. The hamster was anaesthetised by 2 mg of sodium pentobarbital intraperitoneally, and also immunosuppressed by giving cortisone acetate 2-5 mg per 50 g of body weight once every 4 days, starting on the day oftransplanation.
Morphologic and chromosome analyses Approximately 5 x 104 to 1 x 105 cells were inoculated on a square test tube (40 by 17 by 8 mm in size; M A - 8 Miharu) containing a cover glass, 32 by 12 mm, and cultured for 3-7 days. The living cells were observed with an inverted phase-contrast microscope. Cells on a cover slip were also fixed with absolute methanol and stained with 10% Giemsa solution. Periodic Acid Schiff (PAS) and Alcian blue stains were made using twice the normal staining time. For chromosome analysis, the medium was exchanged to one containing Colcemid, 5 x l0 -7 M and the cells were incubated for 1 to 2 hr at 37°C to arrest those in metaphase. The cover slip with the cells was treated by a hypotonic solution, placed carefully in one minute in 0.7% and 0.5% sodium chloride solution and then in 0.2% sodium chloride solution for 20 to 40 min. Then the cells were fixed by passing through 1, 10 and 100% Carnoy solution (3:1 aceto-alcohol) for 1 to 2 rain and finally for 15 min in the fresh 100% Carnoy solution. The specimens were air dried and stained with 10% Giemsa solution (pH 6-8). Three hundred and sixteen of the metaphase cells were observed to calculate the modal ploidy and 25 of the modal range were counted exactly to determine the chromosomal number. The karyotype of the ceils with the modal of chromosomes was carefully analysed in accordance with Denver nomenclature. Cell cultivation for growth curve After being dispersed by a mixed solution of 0.1% trypsin and 0.02°/0 EDTA, the cell suspension containing 2 x l0 s cells was inocu-
RESULTS
Culture course to the establishment The cells were attached well on the bottom of a culture bottle on the first day of the primary culture forming multiple sheets. The culture was apparently successful from the beginning, in contrast to other unsuccessful attempts. The primary culture was grown for 18 days before the first subculture. Since then the cells have been subcultured every 7-14 days, being diluted to three- or four-fold on each passage. The cell proliferation has been maintained except for 4 accidental growth-suppressive periods. It seemed, however, that they happened to be due to inappropriate calf serum, or inappropriate incubation conditions, rather than a characteristic feature of these cancer cells in culture. The line was designated as H E C - 6 (human endometrial cancer--6). When confluent, H E C - 6 cells pile up easily from a monolayer sheet. However, if the cells are not subcultured the sheet often will shed cells and debris into the medium. On the other hand the cells are not easy to trypsinize into single cell suspension, especially in logarithmic growth periods but become easy when confluent.
Human endometrial adenoacanthoma cell-line
Fig. 1. Histology of the original tumor showing adenoacanthoma. Hematoxylin and Eosin (H-E) stain, x 1 5 0 . Fig. 2.
The monolayer-cultured HEC-6 cells reveal ajig-saw puzzle-like or a wheeledpattern. Giemsa stain, x 1 5 0 .
Fig. 3.
The piling-up growth of line HEC 6. Note palisade- or gland-like arrangement. Phase-contrast microscopy, x 1 5 0 .
Fig. 4.
Magnified view of the line showing the cells with anisonucleosis and marked nucleoli. The cytoplasms of which borders are obscure preserve vacuoles. Giemsa stain, x 6 0 0 .
255
256
Hiroyuki Kuramoto and Mieko Hamano
fi~, 7.
A karyotype o f the stem cell showing no apparent structztral aberratiopzs except numerical alteration in groz~p C and F.
Fi~. 8.
Histology
Fig. 9.
Ttze reconstruction (d" a slraltified pacement epithelium i~ also demonslrated on /he other z,ie~'. H E stairl, x 150.
o f the ttmzor zche~ transplanted into adotocarcinoma. H E slniT1, x 150.
a hamster
revealb~g
Human endometrial adenoacanthoma cell-line Morphology
Since the primary culture the cells have remained homogenous and polygonal shaped. No fibroblastic cells have been seen. The arrangement of the actively growing cell sheet appears stream-shaped in polarity, showing a jig-saw puzzle-like or a wheeled pattern (Fig. 2). When the growth progresses H E C - 6 cells grow in multilayers and seemingly reconstruct art original structure obviously forming a palisade-like formation (Fig. 3). Conversely, when the cells grow in a monolayer sheet they proliferate most readily around the cell-free spaces. This seems to be an expression of acinous formation. The cells of the primary culture were classified as malignant columnar cells according to the criteria of a diagnostic exfoliative cytology. They had hyperchromatic nuclei, of varying size, irregular chromatin distribution and one or more large nucleoli. The plasma membrane was ill defined. There was abundant cytoplasm containing many vacuoles (Fig. 4). PAS-positive granules, that were removed by digestion with saliva, were present in the cytoplasm. Alcian blue staining was negative. These morphologic features have been retained by the culture cells. However, the PAS-positive granules disappeared at 30th generation until then they had been a constant feature of the cytoplasm.
257
20--
•
% x
iling time hours
't
E 3
z
I
[
5 Culture
15
doy
Fig. 5. Growth curve of HEC-6 at 37th generation. The population doubling time is calculated as 52 hours.
~1
290 j
n
Rough c o u n f
2n
~3n
4n
5n<
Ploidy E z
Growth kinetics
The growth curve of H E C - 6 cells was examined at 37th passage generation (Fig. 5). The growth showed lag phase, lasting 5 days, followed by a logarithmic growth phase until the l l t h day. Subsequently, the growing rate declined to enter a stationary phase. The population doubling time of the line H E C - 6 during the logarithmic growth phase was 52 hours.
J
I0
43
44
45
Chromosomal
H 46
47
48
49
number
Fig. 6. Distribution of the chromosomal number at the primary culture. The majority of 316 metaphase cells appeared to be on a diploid range by a rough-counting (above) and the exact counting of 25 cells on the diploid mode showed the peak on 46 in number (below).
Chromosome constitution
The chromosome analysis was performed on the primary culture. The modal ploidy of 316 metaphase cells appeared to be obviously diploid by approximate counting. Very few cells deviated from the diploid mode. The exact counting of chromosome number showed the modal peak to be 46 (Fig. 6). The karyotype of the mode was analysed. There was no specific chromosome aberration or marker chromosome (Fig. 7), but irregular increase or decrease of chromosome number in each group was noted. Chromosomes exhibiting slight morphological aberrations were present in almost every group (Table 1). From these
findings, it is deduced that the H E C - 6 line arose from a pseudo-diploid stem cell. Heterotransplantation
The H E C - 6 cells at l lth, 16th and 26th generation respectively were transplanted into the submucosa of a hamster cheek pouch and produced a definite tumour. Histologically in some areas these tumours were typical of adenocarcinoma (Fig. 8). In another field, there appeared to be no gland formation or cells with cytoplasm of a secretory type, but there was construction of a stratified epithelium which was considered as a squamous meta-
Hiroyuki Kuramoto and Mieko Hamano
258
Table 1. Karyotype analysis of HEC-6 on 46 chromosomal number at the primary culture. Variant chromosomes (*) which show minimum morphological aberrations are scattered at random in various groups. The group constitutions in number are altered
No. 1 2 3 4 5 6 7 8
Sketch Chro. No. No. 9 10 11 19 20 24 25 4A
46 46 46 46 46 46 46 46
A1
A2
A3
B
C
D
E
F
G
2 2 3 2 2 2 2 1
3 2+1" 2* 2+ 1" 2 2 2 2
2* 1" 2 + 1" 2 2 2 1+1" 1 + 1"
4 2-t-3" 4 4 4 4 3+1" 3
14 15 14 13 13 15 17 19
7 5 6 5+ 1" 4+ 1" 6 6 5
4 6 6 6 7+2* 6 4+1" 6
6 4 4 4 4 5 4 4
4 4+1" 4 5+ 1" 5 4 4 4
*Variant chromosome.
morphosis (Fig. 9). The findings coincide well with those of the original adenoacanthoma. DISCUSSION
One of the authors was the first to report the established line, H E C - 1 , of h u m a n corpus tumour in 1972, originating from a moderately differentiated adenocarcinoma [1, 2], and mechanism of the progesterone therapy upon endometrial carcinoma was studied by using this line. The suppressive effect on the growth kinetics and the morphological alterations were reported elsewhere [3]. The characteristic features of line H E C - 6 such as the cellular arrangement, diploid modal chromosomal number and heterotransplantability are similar to the HEC-1 line. Conversely, there is the detailed histology of the reconstructed tumour grown as a xenograft and absence of a marker chromosome. Adenoacanthoma of the uterine corpus m a y be defined as a lesion composed of anaplastic glandular element in addition to which there is an epidermoid epithelial component. The latter is usually benign [5]. The histogenesis of the squamous-metaplastic change has been debated. The indifferent or reserve cell, lying between the cylindrical cells and the basement membrane, may give rise to stratified squamous epithelium in response to a variety of stimuli. This might possibly arise in malignant as well as benign endometrial glands. The carcinoma cells may have keen capable of differentiating into squamous epithelium; rather than squamous metaplasia of the normal endometrium, as the result of an inflammatory response from the adjacent carcinoma. The xenograft of the H E C - 6 cells seems to give an answer to this question, since no normal endometrial components
which may transform to a squamous epithelium are present in the graft site. The cultured cell differentiates both adenomatous and squamous elements in an appropriate environment in vivo. The individual cell characteristics determine the malignancy of the lesion. The stem cell of line H E C - 6 is a pseudodiploid. Likewise, various investigators [6-12] have reported, using squash or flame-dry methods, that endometrial cancer has a diploid or near diploid mode. Tseng and Jones [13], and K a t a y a m a and Jones [10] found art entirely normal karyotype in endometrial tumours including an adenoacanthoma which has never been reported in other malignant neoplasms. However, Wagner et al. [14] observed a wide variety of aneuploidy states based on the distribution of nuclear DNA content, and Takemura [6] found polyploid modes in two cases. K a t a y a m a and Jones [10] also stated that in an anaplastic adenocarcinoma and an adenosquamous carcinoma more than half of the cells had hyperdiploid chromosomes. On the other hand, Atkin et al. [15] demonstrated all of the 7 welldifferentiated corpus tumours were diploid or hypodiploid, whereas there were relatively fewer corpus tumours in the tetraploid region and none with DNA values above tetraploid. There may be evidence of a correlation between the histologic grading of malignancy and the extent of chromosome aberrations in the carcinoma of the endometrium, although the majority of the reported cases as well as H E C - 6 gather to a diploid range. A c k n o w l e d g e m e n t s - - A u t h o r s express their gratitude to Prof. Kuniomi Osanai and Prof. Masao ArM for
their criticism and encouragement. This work was supported in part by Grants-in-Aid for Cancer research from the Ministry of Education, Science and Culture, Japan.
Human endometrial adenoacanthoma cell-line REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
H. KURAMOTO,S. TAMURA and Y. NOTAKE,Establishment of a cell line of human endometrial adenocarcinoma in vitro. Amer. J. Obstet. Gynecol. 114, 1012 (1972). H. KURAMOTO, Studies of the growth and cytogenetic properties of human endometrical adenocarcinoma in culture and its development into an established line. Acta obst. gynaec.jap. 19, 47 (1972). H. KURAMOTOand K. SUZUKI,Effects of progesterone on the growth kinetics and the morphology of a human endometrial cancer cell-line. Acta obst. gynaec. jap. 23, 123 (1976). V.J. EVANS,W. R. EARLE,K. K. SANFORD,J. E. SHANNONand H. K. WALTZ, The preparation and handling of replicate tissue cultures for quantitative studies. J. nat. CancerInst. 11,907 (1951). M.S. BAGG:SHand J. D. WOODRUFF,The occurrence of squamous epithelium in the endometrium. Obstet. gynec. Surv,. 22, 69 (1967). T. TAKEMURA, Chromosome survey of normal human endometrium and endometrial carcinoma. Acta obst. gynaec.jap. 7, 300 (1960). R. WAKONIG-VAARTAJA,A human tumour with identifiable cells as evidence for the mutation theory. Brit. J. Cancer 16, 616 (1962). R. WAKONm-VAARTAJA,Chromosomes in gynaecological malignant tumours. Aust. N . Z . J . Obstet. Gynaec. 3, 170 (1963). R. WAKONm-VAARTAJAand D. T. HUGHES, Chromosome studies in 36 gynecological tumours: of the cervix, corpus uteri, ovary, vagina and vulva. Europ. J. Cancer3, 263 (1967). K . P . KATAYAMAand It. W. JONES, Chromosomes of atypical (adenomatous) hyperplasia and carcinoma of the endometrium. Amer. J. Obstet. Gynecol. 97, 978 (1967). M . C . BAKER,A chromosome study of seven near-diploid carcinomas of the corpus uteri. Brit. J. Cancer22, 683 (1968). M . A . STANLEY and J. A. KIRKLA~D, Cytogenetic studies of endometrial carcinoma. Amer. J. Obstet. Gynecol. 11)2, 1070 (1968). P. TSENG and H. W. JONES, Chromosome constitution of carcinoma of the endometrium. Obstet. and Gynec. 33, 741 (1969). D. WAGNER, R. M. RICHART and J. Y. TERNER, Deoxyribonucleic acid content of presumed precursors of endometrial carcinoma. Cancer (Philad.) 20, 2067 (1967). N.B. ATE:Y, B. M. RmHARDSand A. J. Ross, Deoxyribonucleic acid content of carcinoma of the uterus: An assessment of its possible significance in relation to histopathology and clinical course, based on data from 165 cases. Brit. J. Cancer 13, 773 (1959).
259
Establishment and Characterization of the Cell-Line of a Human Edonmetrial Adenoacanthoma HIROYUKI K U R A M O T O and MIEKO HAMANO Department of Obstetrics and Gynecology, and Preventive Medicine System, School of Medicine, Kitasato University, Sagamihara, Japan Abstract A new cell-line of a human endometrial adenoacanthoma, designated as HEC-6, has been established and propagates well with a permanent stability. The population doubling time of the line is calculated as 52 hr. The in vitro morphology reveals the features of a well-differentiated adenocarcinoma such as a jig-saw puzzlelike arrangement and theformation of an acinous cell-free space in the monolayer culture. Each cell consists of a round atypical nucleus with 2 or 3 marked nudeoli and a vacuolated cytoplasm. A piling-up tendency is a prominent feature and forms an obvious palisade-like or glandular structure. The karyotype of the stem cell is a pseudo-diploid in which variant chromosomes occur at random in various groups. When transplanted into a hamster cheek pouch, line HEC-6 demonstrated an adenocarcinoma in addition to a stratified epithelial component. The original characteristics of an adenoacanthoma of the corpus uteri have been maintained in the culture system.
from a 48-year-old patient with carcinoma of the body of the uterus. This nulliparous patient has been well with regular menstrual cycle until she first complained of intermittent bloody discharge for 2 months duration. She used to live in Hiroshima and was 25 when an atomic bomb exploded there during World War II. An endometrial curettage showed an adenoacanthoma of the endometrium and the portion of a squamous epithelium was rather atypical in shape (Fig. 1). The patient was treated by total hysterectomy, bilateral salpingo-oophorectomy and pelvic lymphadenectomy on J u l y 24, 1970. The uterus, the size of a small goose egg, showed a circumscribed tumour on the fundus, bulging into the cavity. The specimen adjacent to the cultured material revealed a well-differentiated adenocarcinoma.
INTRODUCTION
THE ENDOMETRIUM, a target of sex steroid hormones, undergoes a cyclic alteration by the effects of the hormones. It is important to know if the endometrial gland still preserves the ability as a hormone target even after its maligant transformation. The various characteristics of h u m a n endometrial carcin o m a should include the relation to hormones, however, because of the lack of a suitable experimental tool they have yet to be identified in detail. An in vitro cellular system might contribute to these purposes, supplying a convenient research method. We have established line HEC-1 originating from an adenocarcinoma of the corpus uteri and identified various features of the tumour using this in vitro experimental system [1-3]. The present paper reports the establishment of the new corpus tumour cell-line, its morphology, cytogenetic features and heterotransplantability as well as tissue reproducibility.
Culture method A monolayer culture method was used. The tumour material from the excised uterus was first washed twice in a phosphate buffered solution (Ca 2+ and Mg 2+ free) and minced into fragments with fine scissors and ventilated in 0"25% trypsin solution. The fluid and the minced tumour were stirred slowly by a magnetic stirrer (500rev/min) for 15rain.
MATERIAL A N D M E T H O D S Case history of the culture material The fresh tumour material was obtained
Accepted 11 October 1976. 253
254
Hiroyuki Kuramoto and Mieko Hamano
The trypsin solution was changed 4 times. Then the turbid fluid in which the explant materials were dispersed into a single-cell suspension was aspirated and centrifuged twice after mixing with a culture medium. Subsequently, the sediment was resuspended in medium and placed on a stationary culture in 5% carbon dioxide and 95% air or, occasionally, in a closed vessel. Subcultures were made after treating them with solution of 0.1% trypsin and 0"02% ethylendiamine tetraacetate (EDTA).
lated into plastic Petri dishes ( 6 0 m m in diameter; Lux Plastics) and grown as replicate cultures [4] in a humidified atmosphere containing 5% COz and 95% air. Every 2 days after the subculture, two dishes were harvested to count the number of cells. The media in the remaining dishes was changed. The number of cells in each dish was counted 4 times, using a Burker-Tfirk counter plate. The mean was calculated for each dish and for every harvest. The data was plotted as a semilogarithmic graph.
Growth medium Tissue culture medium No. 199 supplemented with 15% calf serum was used, containing penicillin in 100 U/ml and streptomycin in 100 pg/ml.
Transplantation into a hamster A female hamster, 4 weeks old and weighing about 50 grams, was used for the experiment. The cell cultures were dispersed into a suspension of single cells by the trypsin-EDTA solution, and then were washed with culture media and centrifuged twice. The cells, 42.0 or 75-0x 105 in number, were injected into the submucosa of a hamster cheek pouch by a syringe with a No. 22 gauge needle. The hamster was anaesthetised by 2 mg of sodium pentobarbital intraperitoneally, and also immunosuppressed by giving cortisone acetate 2-5 mg per 50 g of body weight once every 4 days, starting on the day oftransplanation.
Morphologic and chromosome analyses Approximately 5 x 104 to 1 x 105 cells were inoculated on a square test tube (40 by 17 by 8 mm in size; M A - 8 Miharu) containing a cover glass, 32 by 12 mm, and cultured for 3-7 days. The living cells were observed with an inverted phase-contrast microscope. Cells on a cover slip were also fixed with absolute methanol and stained with 10% Giemsa solution. Periodic Acid Schiff (PAS) and Alcian blue stains were made using twice the normal staining time. For chromosome analysis, the medium was exchanged to one containing Colcemid, 5 x l0 -7 M and the cells were incubated for 1 to 2 hr at 37°C to arrest those in metaphase. The cover slip with the cells was treated by a hypotonic solution, placed carefully in one minute in 0.7% and 0.5% sodium chloride solution and then in 0.2% sodium chloride solution for 20 to 40 min. Then the cells were fixed by passing through 1, 10 and 100% Carnoy solution (3:1 aceto-alcohol) for 1 to 2 rain and finally for 15 min in the fresh 100% Carnoy solution. The specimens were air dried and stained with 10% Giemsa solution (pH 6-8). Three hundred and sixteen of the metaphase cells were observed to calculate the modal ploidy and 25 of the modal range were counted exactly to determine the chromosomal number. The karyotype of the ceils with the modal of chromosomes was carefully analysed in accordance with Denver nomenclature. Cell cultivation for growth curve After being dispersed by a mixed solution of 0.1% trypsin and 0.02°/0 EDTA, the cell suspension containing 2 x l0 s cells was inocu-
RESULTS
Culture course to the establishment The cells were attached well on the bottom of a culture bottle on the first day of the primary culture forming multiple sheets. The culture was apparently successful from the beginning, in contrast to other unsuccessful attempts. The primary culture was grown for 18 days before the first subculture. Since then the cells have been subcultured every 7-14 days, being diluted to three- or four-fold on each passage. The cell proliferation has been maintained except for 4 accidental growth-suppressive periods. It seemed, however, that they happened to be due to inappropriate calf serum, or inappropriate incubation conditions, rather than a characteristic feature of these cancer cells in culture. The line was designated as H E C - 6 (human endometrial cancer--6). When confluent, H E C - 6 cells pile up easily from a monolayer sheet. However, if the cells are not subcultured the sheet often will shed cells and debris into the medium. On the other hand the cells are not easy to trypsinize into single cell suspension, especially in logarithmic growth periods but become easy when confluent.
Human endometrial adenoacanthoma cell-line
Fig. 1. Histology of the original tumor showing adenoacanthoma. Hematoxylin and Eosin (H-E) stain, x 1 5 0 . Fig. 2.
The monolayer-cultured HEC-6 cells reveal ajig-saw puzzle-like or a wheeledpattern. Giemsa stain, x 1 5 0 .
Fig. 3.
The piling-up growth of line HEC 6. Note palisade- or gland-like arrangement. Phase-contrast microscopy, x 1 5 0 .
Fig. 4.
Magnified view of the line showing the cells with anisonucleosis and marked nucleoli. The cytoplasms of which borders are obscure preserve vacuoles. Giemsa stain, x 6 0 0 .
255
256
Hiroyuki Kuramoto and Mieko Hamano
fi~, 7.
A karyotype o f the stem cell showing no apparent structztral aberratiopzs except numerical alteration in groz~p C and F.
Fi~. 8.
Histology
Fig. 9.
Ttze reconstruction (d" a slraltified pacement epithelium i~ also demonslrated on /he other z,ie~'. H E stairl, x 150.
o f the ttmzor zche~ transplanted into adotocarcinoma. H E slniT1, x 150.
a hamster
revealb~g
Human endometrial adenoacanthoma cell-line Morphology
Since the primary culture the cells have remained homogenous and polygonal shaped. No fibroblastic cells have been seen. The arrangement of the actively growing cell sheet appears stream-shaped in polarity, showing a jig-saw puzzle-like or a wheeled pattern (Fig. 2). When the growth progresses H E C - 6 cells grow in multilayers and seemingly reconstruct art original structure obviously forming a palisade-like formation (Fig. 3). Conversely, when the cells grow in a monolayer sheet they proliferate most readily around the cell-free spaces. This seems to be an expression of acinous formation. The cells of the primary culture were classified as malignant columnar cells according to the criteria of a diagnostic exfoliative cytology. They had hyperchromatic nuclei, of varying size, irregular chromatin distribution and one or more large nucleoli. The plasma membrane was ill defined. There was abundant cytoplasm containing many vacuoles (Fig. 4). PAS-positive granules, that were removed by digestion with saliva, were present in the cytoplasm. Alcian blue staining was negative. These morphologic features have been retained by the culture cells. However, the PAS-positive granules disappeared at 30th generation until then they had been a constant feature of the cytoplasm.
257
20--
•
% x
iling time hours
't
E 3
z
I
[
5 Culture
15
doy
Fig. 5. Growth curve of HEC-6 at 37th generation. The population doubling time is calculated as 52 hours.
~1
290 j
n
Rough c o u n f
2n
~3n
4n
5n<
Ploidy E z
Growth kinetics
The growth curve of H E C - 6 cells was examined at 37th passage generation (Fig. 5). The growth showed lag phase, lasting 5 days, followed by a logarithmic growth phase until the l l t h day. Subsequently, the growing rate declined to enter a stationary phase. The population doubling time of the line H E C - 6 during the logarithmic growth phase was 52 hours.
J
I0
43
44
45
Chromosomal
H 46
47
48
49
number
Fig. 6. Distribution of the chromosomal number at the primary culture. The majority of 316 metaphase cells appeared to be on a diploid range by a rough-counting (above) and the exact counting of 25 cells on the diploid mode showed the peak on 46 in number (below).
Chromosome constitution
The chromosome analysis was performed on the primary culture. The modal ploidy of 316 metaphase cells appeared to be obviously diploid by approximate counting. Very few cells deviated from the diploid mode. The exact counting of chromosome number showed the modal peak to be 46 (Fig. 6). The karyotype of the mode was analysed. There was no specific chromosome aberration or marker chromosome (Fig. 7), but irregular increase or decrease of chromosome number in each group was noted. Chromosomes exhibiting slight morphological aberrations were present in almost every group (Table 1). From these
findings, it is deduced that the H E C - 6 line arose from a pseudo-diploid stem cell. Heterotransplantation
The H E C - 6 cells at l lth, 16th and 26th generation respectively were transplanted into the submucosa of a hamster cheek pouch and produced a definite tumour. Histologically in some areas these tumours were typical of adenocarcinoma (Fig. 8). In another field, there appeared to be no gland formation or cells with cytoplasm of a secretory type, but there was construction of a stratified epithelium which was considered as a squamous meta-
Hiroyuki Kuramoto and Mieko Hamano
258
Table 1. Karyotype analysis of HEC-6 on 46 chromosomal number at the primary culture. Variant chromosomes (*) which show minimum morphological aberrations are scattered at random in various groups. The group constitutions in number are altered
No. 1 2 3 4 5 6 7 8
Sketch Chro. No. No. 9 10 11 19 20 24 25 4A
46 46 46 46 46 46 46 46
A1
A2
A3
B
C
D
E
F
G
2 2 3 2 2 2 2 1
3 2+1" 2* 2+ 1" 2 2 2 2
2* 1" 2 + 1" 2 2 2 1+1" 1 + 1"
4 2-t-3" 4 4 4 4 3+1" 3
14 15 14 13 13 15 17 19
7 5 6 5+ 1" 4+ 1" 6 6 5
4 6 6 6 7+2* 6 4+1" 6
6 4 4 4 4 5 4 4
4 4+1" 4 5+ 1" 5 4 4 4
*Variant chromosome.
morphosis (Fig. 9). The findings coincide well with those of the original adenoacanthoma. DISCUSSION
One of the authors was the first to report the established line, H E C - 1 , of h u m a n corpus tumour in 1972, originating from a moderately differentiated adenocarcinoma [1, 2], and mechanism of the progesterone therapy upon endometrial carcinoma was studied by using this line. The suppressive effect on the growth kinetics and the morphological alterations were reported elsewhere [3]. The characteristic features of line H E C - 6 such as the cellular arrangement, diploid modal chromosomal number and heterotransplantability are similar to the HEC-1 line. Conversely, there is the detailed histology of the reconstructed tumour grown as a xenograft and absence of a marker chromosome. Adenoacanthoma of the uterine corpus m a y be defined as a lesion composed of anaplastic glandular element in addition to which there is an epidermoid epithelial component. The latter is usually benign [5]. The histogenesis of the squamous-metaplastic change has been debated. The indifferent or reserve cell, lying between the cylindrical cells and the basement membrane, may give rise to stratified squamous epithelium in response to a variety of stimuli. This might possibly arise in malignant as well as benign endometrial glands. The carcinoma cells may have keen capable of differentiating into squamous epithelium; rather than squamous metaplasia of the normal endometrium, as the result of an inflammatory response from the adjacent carcinoma. The xenograft of the H E C - 6 cells seems to give an answer to this question, since no normal endometrial components
which may transform to a squamous epithelium are present in the graft site. The cultured cell differentiates both adenomatous and squamous elements in an appropriate environment in vivo. The individual cell characteristics determine the malignancy of the lesion. The stem cell of line H E C - 6 is a pseudodiploid. Likewise, various investigators [6-12] have reported, using squash or flame-dry methods, that endometrial cancer has a diploid or near diploid mode. Tseng and Jones [13], and K a t a y a m a and Jones [10] found art entirely normal karyotype in endometrial tumours including an adenoacanthoma which has never been reported in other malignant neoplasms. However, Wagner et al. [14] observed a wide variety of aneuploidy states based on the distribution of nuclear DNA content, and Takemura [6] found polyploid modes in two cases. K a t a y a m a and Jones [10] also stated that in an anaplastic adenocarcinoma and an adenosquamous carcinoma more than half of the cells had hyperdiploid chromosomes. On the other hand, Atkin et al. [15] demonstrated all of the 7 welldifferentiated corpus tumours were diploid or hypodiploid, whereas there were relatively fewer corpus tumours in the tetraploid region and none with DNA values above tetraploid. There may be evidence of a correlation between the histologic grading of malignancy and the extent of chromosome aberrations in the carcinoma of the endometrium, although the majority of the reported cases as well as H E C - 6 gather to a diploid range. A c k n o w l e d g e m e n t s - - A u t h o r s express their gratitude to Prof. Kuniomi Osanai and Prof. Masao ArM for
their criticism and encouragement. This work was supported in part by Grants-in-Aid for Cancer research from the Ministry of Education, Science and Culture, Japan.
Human endometrial adenoacanthoma cell-line REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
H. KURAMOTO,S. TAMURA and Y. NOTAKE,Establishment of a cell line of human endometrial adenocarcinoma in vitro. Amer. J. Obstet. Gynecol. 114, 1012 (1972). H. KURAMOTO, Studies of the growth and cytogenetic properties of human endometrical adenocarcinoma in culture and its development into an established line. Acta obst. gynaec.jap. 19, 47 (1972). H. KURAMOTOand K. SUZUKI,Effects of progesterone on the growth kinetics and the morphology of a human endometrial cancer cell-line. Acta obst. gynaec. jap. 23, 123 (1976). V.J. EVANS,W. R. EARLE,K. K. SANFORD,J. E. SHANNONand H. K. WALTZ, The preparation and handling of replicate tissue cultures for quantitative studies. J. nat. CancerInst. 11,907 (1951). M.S. BAGG:SHand J. D. WOODRUFF,The occurrence of squamous epithelium in the endometrium. Obstet. gynec. Surv,. 22, 69 (1967). T. TAKEMURA, Chromosome survey of normal human endometrium and endometrial carcinoma. Acta obst. gynaec.jap. 7, 300 (1960). R. WAKONIG-VAARTAJA,A human tumour with identifiable cells as evidence for the mutation theory. Brit. J. Cancer 16, 616 (1962). R. WAKONm-VAARTAJA,Chromosomes in gynaecological malignant tumours. Aust. N . Z . J . Obstet. Gynaec. 3, 170 (1963). R. WAKONm-VAARTAJAand D. T. HUGHES, Chromosome studies in 36 gynecological tumours: of the cervix, corpus uteri, ovary, vagina and vulva. Europ. J. Cancer3, 263 (1967). K . P . KATAYAMAand It. W. JONES, Chromosomes of atypical (adenomatous) hyperplasia and carcinoma of the endometrium. Amer. J. Obstet. Gynecol. 97, 978 (1967). M . C . BAKER,A chromosome study of seven near-diploid carcinomas of the corpus uteri. Brit. J. Cancer22, 683 (1968). M . A . STANLEY and J. A. KIRKLA~D, Cytogenetic studies of endometrial carcinoma. Amer. J. Obstet. Gynecol. 11)2, 1070 (1968). P. TSENG and H. W. JONES, Chromosome constitution of carcinoma of the endometrium. Obstet. and Gynec. 33, 741 (1969). D. WAGNER, R. M. RICHART and J. Y. TERNER, Deoxyribonucleic acid content of presumed precursors of endometrial carcinoma. Cancer (Philad.) 20, 2067 (1967). N.B. ATE:Y, B. M. RmHARDSand A. J. Ross, Deoxyribonucleic acid content of carcinoma of the uterus: An assessment of its possible significance in relation to histopathology and clinical course, based on data from 165 cases. Brit. J. Cancer 13, 773 (1959).
259