Establishment and characterization of a highly tumorigenic human diploid endometrial cancer cell line

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Establishment and Characterization of a Highly Tumorigenic Human Diploid Endometrial Cancer Cell Line Jin-Woo Kim, Chun-Geun Lee, Soo-Kyung Choi, Jae-Hoon Kim, Tae-Eung Kim, Joon-Mo Lee, Jong-Gu Rha, and Sung-Eun Namkoong ABSTRACT: A n e w cell line designated CUME-1 has been established from a poorly differentiated

endometrial adenocarcinoma of the uterus. This cell line grew well without interruption for more than 88 months and 110 serial passages were successively carried out. The cells were highly tumorigenic in nude mice (85%). Repeated karyotype analyses from early (4th) to late (55th) passages of this ceil line revealed a diploid stable clone in each passages without any noticeable structural or numerical aberrations. But from the 80th passage, a subpopulation with reciprocal translocation between chromosomes lq and 9q consistently appeared and was observed in about 30% of the cells. This cell line is one of the rare examples of experimentally proved tumorigenic cells of human solid tumor origin that retains the diploid karyotype in vitro. HLA typing indicated the presence of DR4, DR13, DQ3, and DQ6. Cytosol estrogen and progesterone receptors were found both in fresh primary tumor and in this cell line. Gonadotropin-releasing hormone (Gn-RH) receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in cultured cells. Using the single-strand conformation polymorphism (SSCP) technique, we have screened CUME-1 ceils for p53 mutation in exons 4 to 9. No mobility shift was observed. This cell line may be useful in studying the in vitro chromosomal evolution of the cell line and the in rive properties of human endometrial adenocarcinoma. Q Elsevier Science Inc., 1997

INTRODUCTION

There is a geographic correlation in the frequency of e n d o m e t r i a l cancer, w i t h a low rate of this cancer in Asiatic p o p u l a t i o n s and a high rate in North A m e r i c a n s [1]. But, the overall increase in the i n c i d e n c e of e n d o m e t r i a l carcinoma in the past decade in Korea has been alarming. In the early nineties a 3-fold increase in i n c i d e n c e has been reported [2]. Because life e x p e c t a n c y among females is increasing and because e n d o m e t r i a l c a r c i n o m a is rising in incidence, it is m a n d a t o r y to elucidate the nature of this neoplasm. F r o m this p o i n t of view, it is necessary to establish m a n y cell lines from e n d o m e t r i a l carcinoma, and to use t h e m for

From the Department of Obstetrics and Gynecology, Kangnam St. Mary's Hospital, Catholic Cancer Center, Catholic University Medical College (].-W. K., J.-H. K., T.-E. K., J.-M. L., J.-G. R., S.-E. N.), and Department of Medical Genetics, Hanyang University School of Medicine (C.-G. L.), and Genetic Research Laboratory, Cheil General Hospital (S.-K. C.), Seou], South Korea. Address reprint requests to: Jin-Woo Kiln, M.D., Department of Obstetrics and Gynecology, Kangnam St. Mary's Hospital, Catholic University Medical College, 505 Banpo-dong, Seocho-ku, Seoul, 137-040, Korea. Received July 11, 1996; accepted October 18, 1996. Cancer Genet Cytogenet 99:1-10 (1997) © Elsevier Science Inc., 1997 655 Avenue of the Americas, N e w York, NY 10010

e x a m i n a t i o n of in vitro experiments and for d e v e l o p m e n t of n e w therapeutic approaches. But, to date, a limited number of lines has been established from h u m a n e n d o m e t r i a l a d e n o c a r c i n o m a [3-9]. Unlike the other solid tumors, the majority of the reported cases of e n d o m e t r i a l cancer have a distinct m o d e in the d i p l o i d range with m i n i m a l c h r o m o s o m a l abnormalities [10, 111. It is more favorable for the identification of specific c h r o m o s o m a l or m o l e c u l a r changes than the other solid tumors because these abnormalities are not obscured by other n u m e r o u s karyotypic changes. However, as for an in vitro e x p e r i m e n t a l model, only one tumorigenic cell line with n o r m a l karyotype from e n d o m e t r i a l cancer was described up to date [12]. But it is uncertain w h e t h e r the tumorigenicity or the other nature of the t u m o r cells could be m a i n t a i n e d in the long-term culture, w h i c h u s u a l l y resulted in the c h r o m o s o m a l evolution. In the present study, we established a highly tumorigenic e n d o m e t r i a l cancer cell line with a normal d i p l o i d karyotype and tested the stability of the cell line by sequential karyotypic analyses and tumorigenic assay. This cell line has also been analyzed w i t h respect to the in vitro growth properties, cellular ultrastructure, isozyme patterns, HLA typing, hormone and tumor marker secretions, steroid

0165-4608/97/$17.00 PII S0165-4608(96)00389-5

2 hormone receptor existences, Gn-RH receptor mRNA expression, and p53 tumor suppressor gene mutation.

MATERIALS AND METHODS Origin of the Cell line A 58-year-old Korean woman presented with endometrial carcinoma of the uterus, and was referred to the Department of Gynecology of Catholic University Medical Hospital where the diagnosis of endometrial adenocarcinoma FIGO [13] Stage IA Grade 3 was made. She underwent a modified radical hysterectomy (Wertheim operation) with pelvic and para-aortic lymphadenectomy on June 10, 1988. At the time of surgery she was found to have an extensive endometrial carcinoma invading the partial thickness of the myometrium in the fundal portion. This cell line was derived from a cancerous bulky mass obtained from the operation materials for primary culture. All of the resected lymph nodes were negative. To date the patient is still alive without any signs of recurrence.

Establishment of the Cell Line Specimens were held in ice-cold Waymouth's MB 752/1 medium (GIBCO, Grand Island, NY) supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 ixg/ml streptomycin, and 20% fetal bovine serum (FBS). After mechanical dissociation, tumor fragments and cellular suspensions were collected, washed 3 times, and finally centrifuged. The sediments were resuspended in medium and seeded into 25-cm 2 tissue culture flasks to initiate in vitro growth of the cells. Routine assays for mycoplasma using a Geneprobe rapid detection system (Gene-probe, San Diego, CA) were negative.

Morphology of the Cultured Cells The cells grown in culture flasks were photographed by phase-contrast microscopy and cells grown on slides were washed with phosphate-buffered saline (PBS) and fixed in 95% ethanol. They were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), mucicarmine, and Alcian blue. For transmission electron microscopic study, the tumor cells were seeded on a tissue culture chamber slide (Lab-Tek, Naperville, IL), fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. They were then postfixed with a 2% osmium tetroxide. Specimens were dehydrated through an ascending series of ethanol, and infiltrated with pure Epon 812 overnight. After polymerization, thin sections were contrasted with uranyl acetate and lead citrate before being examined with a JEOL 1200 EX electron microscope.

Growth Characteristics Growth curve was constructed by seeding 1 x 105 cells into 25-cm 2 flasks and incubated for 11 days. The population doubling time was calculated from the growth curve. For studies of plating efficiency, one hundred tumor cells were plated in each of the ten 25-cm 2 flasks. After two weeks, the colonies formed were fixed in 95% ethanol and stained with 0.5% crystal violet in 25% ethanol. The number of colonies with more than 50 cells each was counted.

J.-W. Kim et al.

Tumor Xenografts Tumorigenicity was tested in the 30th passage using 40 mice of 4- to 6-week-old female nude mice (athymic nu/nu on BALB/c background). Five × 10 ~ cells were injected subcutaneously into the posterior lateral aspect of the trunk (lumbar region) of 30 mice. One × 105 cells were also injected into the lateral tail veins of the other 10 mice. Five × 1 0 6 cells were also injected subcutaneously in the 100th passage to compare the tumorigenicity with the 30th passage. Only the cell suspensions with greater than 95% viability, as assessed by trypan blue dye exclusion, were used for injection.

Isozyme Analysis Genetic signature analysis was conducted on CUME-1 cells. Electrophoretic phenotypes were determined for the products of the following 3 enzyme loci: glucose 6-phosphate dehydrogenase (G6PD), lactic dehydrogenase (LDH), and phosphoglucomutase (PGM) 1 by horizontal electrophoresis. HeLa cells were typed for G6PD as a control for the type A phenotype.

Cytogenetic Analysis Repeated chromosome analyses were carried out at early to late passages (4th, 55th, and 80th) to examine the in vitro chromosomal evolution of this cell line. A total of 30 metaphase spreads in each passage were photographed and the chromosome numbers in each spread were counted. Harvesting, fixation, and G-banding of the chromosomes were induced by using routine techniques published elsewhere [14]. To reveal the exact origin of the translocated marker chromosome, we performed fluorescence in sitn hybridization (FISH) with painting probes (ChromoprobesM 1 and 9: Cytocell, Applied Imaging Co., UK) according to the protocols supplied by the company, and analyzed the karyotype with Cytovision (Applied Imaging Co, UK).

HLA Typing HLA typing was performed on cultured cells using allelespecific PCR genotyping method [15]. The sequences of the locus-specific primers and allele-specific primers were designed according to Marsh and Bodmer [16]. HLA genotyping for three of the polymorphic class II loci (DRB1, DQA1, and DQB1) was conducted using two-step PCR. Each reaction mixture contained 0.1 pxg cellular DNA, 0.125 mM of each dNTP, 0.2 U Taq polymerase (Boehringer-Mannheim, Germany), and 15 pmol of each locusspecific primer. First amplification was carried out for 31 cycles. The locus specifically amplified products were analyzed on 1.5% agarose gel and the reaction products were reamplified for 16 cycles with allele-specific primer.

Hormone Receptor Assay The cultured cells were analyzed for estrogen receptor (ER) and progesterone receptor (PR). For the ER and PR assay, Rianen [3H] Estrogen and Progestin Receptor Assay kits (Du Pont Co., Billerica, MA) were used, respectively. The data, after correction for nonspecific binding, were

Establishment a n d Characterization of a Cancer Cell Line

a n a l y z e d using a Scatchard plot to d e t e r m i n e the n u m b e r of sites. Receptors were c o n s i d e r e d absent if less than 9 fmole ER or PR were present per milligram of protein.

Gn-RH Receptor nRNA Expression Total RNA was extracted from CUME-1 cells, using a commercial system (RNeasy total RNA kit) p r o v i d e d by Qiagen (Qiagen Inc., Germany). RT-PCR was carried out using RNA PCR kit (Perking Elmer, NJ) u n d e r the conditions reco m m e n d e d by the supplier. Reverse transcripted p r o d u c t (20 txl) was a m p l i f i e d in a 100-1xl reaction containing 10

3

m M Tris HC1 (pH 8.3), 50 m M KC1, 2.5 m M MgC12, 50 txM of each dNTP, 2.5 U AmpliTaq DNA p o l y m e r a s e (Perkin Elmer, NJ), and 100 p m o l of each primer. The sequences of oligonucleotide primers, u p s t r e a m (5'-GACCTTGTCTGGAAAGATCC-3') a n d d o w n s t r e a m p r i m e r (5'-CAGGCTGATCACCACCATCA-3'), were synthesized according to the p u b l i s h e d h u m a n Gn-RH receptor cDNA sequence [17]. We carried out 35 cycles of amplification: denaturation at 95°C for 20 seconds, annealing-extension at 60°C for 30 seconds, followed by a final extension for 7 min at 72°C. The DNA p r o d u c t (10 txl) was run on 2% agarose gel,

Figure I (A) Histology of the original endometrial adenocarcinoma, showing poorly differentiated carcinoma. Under a light microscope, a large solid mass with infrequent glandular lumina and focal necrosis was found (hematoxylin and eosin, ×200). (B) Histology of subcutaneous tumor nodules taken from nude mice showed large solid masses composed of poorly differentiated carcinoma cells that closely resembled the original tumor (hematoxylin and eosin, x200).

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Table 1

Tumorigenicity of CUME-1 cells in n u d e mice

Passage number

Number of cells injected

Injection site

Number of animals

Died unexpectedly

Subcutaneous tumor formation

Lung colonies

30 30 100

5 x 106 I × 105 5 × 106

scc~ ivb sC'

30 10 30

3 1 5

23 of 27 (85%) 22 of 25 (88%)

2 of 23 (9%) 5 of 9 (56%) 2 of 22 (9%)

"Subcutaneousinoculation. J'Iutravenousinoculation.

and bands were visualized by e t h i d i u m bromide staining on an ultraviolet transilluminator. One h u n d r e d twenty three bp ladder DNA (Gibco-BRL) was used as a size marker. H u m a n ovarian carcinoma cell line, SK-OV3, was used as a positive control for Gn-RH receptor mRNA expression [18]. The predicted fragment amplified by PCR was 319 bp.

Analysis of p53 Gene by PCR-SSCP To search for p53 gene mutations by the PCR-SSCP method [19], four primer sets encompassing so-called "mutational hot spots," exon 4, exons 5 and 6, exon 7, and exons 8 a n d 9 were designed according to previously published sequences [20]. Extracted DNA was purified with Gene Clean II kit (Bio 101 Inc., La Jolla, CA) for subsequent PCR. Each reaction mixture (5 p~l) contained 50 ng DNA, 1.5 mM MgC12, 50 ~M of each dNTP, 0.037 MBq [~-32p]dCTP (Amersham), 0.125 U Taq polymerase, and 2 pmol of each primer. Amplification was carried out for 35 cycles of 30 seconds at 95°C and 2 m i n at (Tm-5)°C. Three microliters of reaction mixtures was then analyzed on 1.5% agarose gel containing ethidium bromide. The remaining 2 ~l of

mixture was diluted 50-fold with formamide dye solution (95% formamide, 20 mM EDTA, 0.05% b r o m o p h e n o l blue, and 0.05% xylene cyanole FF) followed by denaturation by boiling. Two microliters of each sample were electrophoresed on a 6% polyacrylamide gel containing 5% glycerol and the gels were exposed to Xray film for autoradiography for 12 to 24 hours at -70°C. DNA from normal peripheral blood lymphocytes was used as a negative control.

RESULTS Morphology of the Original Tumor Histologic examination of the original tumor specimen showed an adenocarcinoma of the e n d o m e t r i u m that was classified as poorly differentiated. Under a light microscope, a solid mass with infrequent glandular l u m i n a and focal necrosis was seen (Fig. 1A).

Establishment of the Cell Line The CUME-1 cell culture was started on June 10, 1988. After 21 days in the stationary period, favorable outgrowth

Figure 2 Phase-contrast

features of the monolayer-cultured CUME-1 cells revealing a sheet of polygonal ceils with a compact arrangement. Scattered round ceils are surrounded by polygonal cells. Vesicular nuclei and prominent nucleoli were seen. (×100.)

Establishment and Characterization of a Cancer Cell Line

F i g u r e 3 Transmission electron micrographs illustrating representative characteristics of tissue culture cells. (A) Cultured cells show high nucleus-cytoplasmic ratio, euchromatic nuclei, prominent nucleoli, and well-developed microvilli. (B) Higher magnification shows desmosomes (arrows), well-developed numerous rough endoplasmic reticula (open arrows) and polysomes (arrowheads), intracytoplasmic lipid droplets (L), and mitochondria (M). Scale bar, 1 ~m.

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was found, but no contamination by fibroblasts. As the cells became more dense, contact inhibition was not observed and the cells frequently overlapped one another. The CUME-1 cells grew well without interruption for more than 88 months and 110 serial passages were successively carried out.

were observed, and rough endoplasmic reticulums and polysomes were numerous and well-developed (Fig. 3B).

Growth Characteristics Analysis of the growth curve of the CUME-1 cells at passage 100 showed that population doubling time was approximately 56 hours and the plating efficiency was 28%.

Tumorigenicity and Metastasis The subcutaneous injection of cultured cells into nude mice gave rise to fast growing tumors. Following subcutaneous injection, tumors developed in 23 of 27 mice (85%) after the inoculation (3 mice died unexpectedly) in the 30th passage (Table 1). The tumor nodules reached to a size of approximately 1 cm in diameter within 3 weeks. The transplanted subcutaneous tumors closely resembled the original tumor (Fig. 1B). Of 23 mice with tumors that were killed after subcutaneous injection, 2 (9%) were found to have microscopic lung metastases. Following intravenous injection into the lateral tail vein, 5 of 9 mice (56%) had evidence of pulmonary metastasis at the time of termination in the 30th passage (Table 1). Subcutaneous tumor formations were also observed in 22 of 25 mice (88%) in the 100th passage (Table 1). As a result, there were no distinct differences between two passages (the 30th passage and the lOOth passage) following subcutaneous injection.

Morphology of the Cultured Cells Figure 2 is a phase-contrast micrograph of the cultured CUME-1 cells. In the monolayer cultures there were polygonal-shaped cells in a pavement-like arrangement, piled up without contact inhibition. The cytoplasms were stained positively by PAS and mucicarmine, and negatively with Alcian blue. The epithelial nature of the cultured cells was confirmed by ultrastructural analysis. An electron micrograph of the CUME-1 cells showed the presence of desmosomes (Fig. 3A). Intracytoplasmic lipid droplets

2

4

Isozyme Analysis The G6PD electrophoretic pattern of CUME-1 cells showed type B phenotype. The analysis of the LDH isozyme revealed five bands (LDH1, LDH2, LDH3, LDH4, and LDH5) with mobilities characteristic of human tissue. The phenotype of PGM1 of this cell line was identical to those of HeLa (data not shown).

Cytogenetic Analysis From the 4th to the 55th passage, all the metaphases analyzed were normal diploid karyotypes. No noticeable numerical and structural abnormalities were detected with conventional karyotype analysis. From the 80th passage, a subpopulation with one marker chromosome of reciprocal translocation between chromosomes 1 and 9 consistently appeared and was observed in about 30% of cells (Fig. 4). The exact origin of the translocated chromosome was confirmed by FISH using painting probes for chromosomes 1 and 9 (Fig. 5). The high resolution karyotyping revealed that the breakpoints of the marker chromosome were lq21.1 and 9q34.1 (Fig. 6). No significant minor changes of other chromosomes were observed in the detailed examination with high resolution karyotyping at both the 4th and 55th passage (data not shown). HLA Typing HLA class II phenotypes were DR4, DR13, DQ3, and DQ6, and HLA class II allele specificities were DRBI*04**,

S!!~

!) )l t,c K 13

14

tl 1~

1G

,, 22

|¢ x

Sex Chrom~o

¥

Sex nle~

Chl~nIGs~mos

Figure 4 Representative G-banded karyotype of CUME-1, showing the t(1;9).

Establishment a n d Characterization of a Cancer Cell Line

7

A

Figure 5 Fluorescence in situ hybridization with the painting probes of chromosomes 1 (A) and 9 (B) of the CUME-1 cells containing t(1;9)

DRB1*13**, DQAI*03**, DQAI*0101, DQBI*03**, and DQBI*06**.

Hormone Receptors ER a n d PR were found in the p a t i e n t ' s tumor. Cytosol ER (17 fmol/mg protein) and PR (15 fmol/mg protein) were also present in the c u l t u r e d CUME-1 cells.

Gn-RH Receptor mRNA Expression PCR a m p l i f i c a t i o n of first-strand cDNA from CUME-1 and SK-OV3 cells was c o n d u c t e d w i t h an oligonucleotide primer set. Gn-RH receptor mRNA was detected in CUME-1 cells. SK-OV3 cells also gave a p r e d o m i n a n t p r o d u c t (319 bp) identical to that obtained in CUME-1 cells as s h o w n in Figure 7.

PCR-SSCP Analysis of the p53 Gene. DNA extracted from CUME-1 cells was used for amplification of exons 4 to 9 of p53 gene and this PCR p r o d u c t was a n a l y z e d using the SSCP method. No m o b i l i t y shift was observed in this PCR p r o d u c t (data not shown).

DISCUSSION This cell line exhibited a relatively high potential of tumorigenicity, i.e., more than 85% tumorigenic to nude mice following subcutaneous injection, and the implanted nude mice tumors showed the characteristic histology of poorly differentiated endometrial adenocarcinoma. Along with their tumorigenicity, spontaneous metastasis to lung was observed in sacrificed animals after subcutaneous injection of cells, and experimental metastasis to lung was also observed after

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J.-W. Kim et el.

I t ( I ;9)(q2 I . I ;q 34. I )

intravenous injection of cells. Easy tranplantability and reproducibility of the original in vivo characteristics make this cell line a valuable experimental system for the study of in vivo properties of h u m a n endometrial adenocarcinoma.

Figure 7 Polymerase chain reaction amplification of first-strand cDNA from CUME-1 (lane 1), and SK-OV3 (lane 2) cells. Oligonucleotide primers, upstream and downstream (5'-GACCTTGTCTGGAAAGATCC-3', and 5'-CAGGCTGATCACCACCATCA-3') were used.

1 2

319

9

Figure 6 High resolution partial karyotype with idiogram showing the breakpoints involved in the t(1;9).

Both diploid and aneuploid karyotypes are often detected in the same primary cultures derived from solid tumor tissues. However, these diploid cells invariably senesce, fail to grow in athymic or i m m u n o s u p p r e s s e d experimental animals, and p r e s u m a b l y are originated from surrounding n o r m a l tissues of the tumor biopsies. Thus far, the d i p l o i d karyotypes are extremely rare in t u m o r cell lines, w h i c h are especially established from solid tumors. There is only one report of a n o r m a l d i p l o i d cell line, SK-UT-1B, w h i c h was established from uterine m i x e d m e s o d e r m a l tumor [12]. This cell line d e m o n s t r a t e d a high tumorigenicity in vivo like CUME-1 cells. Some breast cancer cells with diploid karyotypes were reported to have invasiveness to amniotic extracellular matrix [21, 22], but it was uncertain that these cell lines still have the tumorigenicity in vivo. It is suggested that the karyotypes of the cultured cells m a y undergo considerable changes in vitro and some of these changes m a y represent the malignant transformation process of the t u m o r cells in vivo [23, 24]. CUME-1 cells also demonstrated structural instability in the later passages, but it is interesting to note that the karyotypic change of this cell line involves only very specific regions of c h r o m o s o m e s 1 and 9. So, this cell line is a rare example having a unique c h r o m o s o m a l change in the long-term culture because most of the sequential analyses of the other cell lines d e m o n s t r a t e d gross p l o i d y change or acc o m p a n i e d the r a n d o m n u m e r i c a l or structural anomalies [24-27]. The c h r o m o s o m e 1 anomalies especially including the region of the long arm were reported in most of the malignant tumors [28] and their frequent occurrences in carcinoma of e n d o m e t r i u m have been described in several reports [10, 11]. The anomalies of the chromosome 9 showing various breakpoints in the long arm region of the e n d o m e t r i a l carcinoma were also reported [11]. It is noticeable that the similar reciprocal translocation b e t w e e n c h r o m o s o m e s 1 and 9 as in our CUME-1 cells was described in the primary adenocarcinoma of the endometrium

Establishment and Characterization of a Cancer Cell Line

[11]. Thus, we suggest the late change of this cell line may represent one of the c o m m o n chromosome changes in malignant transformation of the endometrial cancer. However, the u n d e r l y i n g m e c h a n i s m s in gaining growth advantage of the cells with translocation over the normal diploid cells r e m a i n as a task to be solved. This cell line is peculiar in the sense of high tumorigenicity with normal diploid karyotype u n t i l the 55th passage. Thus, it may be useful as a good experimental model for the investigations of molecular changes w i t h o u t apparent chromosomal aberrations. It will be especially useful for the identification of cancer-relevant gene or genes for tumorigenicity in endometrial cancer by the method of differential display of mRNA techniques compared with normal diploid endometrial cells or cell lines. Additionally, the specific chromosomal change in the later passages of this cell line may add to the usefulness for the study of chromosomal evolution and for the isolation of specific genes located in the long arms of the chromosomes 1 and 9. Because most of endometrial carcinoma cell lines were derived from poorly differentiated tumors, endometrial carcinoma cell lines are mostly h o r m o n e receptor-negative [4, 7]. It is generally believed that poorly differentiated and recurrent endometrial carcinomas often lack steroid receptors and exhibit a poor response to progestin. But, CUME-1 cell line derived from a poorly differentiated tumor, such as EC-MZ-3 cells [9] showed h o r m o n e receptorpositive. The original t u m o r of cell line CUME-1 also had positive h o r m o n e receptors. Therefore, the establishment of a receptor-positive poorly differentiated endometrial cancer cell line w o u l d be a useful experimental system i n the study of receptor expression and growth regulation by steroid hormones in the e n d o m e t r i u m . The a n t i t u m o r action of Gn-RH analog in breast and prostate cancers has been s h o w n to result from a desensitization or down-regulation of Gn-RH receptors in the pituitary, with a c o n s e q u e n t decline in gonadotropin secretion and gonadal steroid p r o d u c t i o n [29]. But there are indications that Gn-RH analog suppresses the growth of the cancer cells in vitro and that the specific binding sites for Gn-RH are demonstrated in certain tumors responsive to the GnRH analog [30]. These findings suggest direct regulatory effects of Gn-RH on the tumor growth of certain Gn-RHsensitive cancers [31]. Because the sensitivity of tumors to h o r m o n a l control is associated with the possession of receptors for the hormone, the expression of Gn-RH receptor in this cell line may provide a rationale for the further exploration of a direct antitumor effect of the Gn-RH analog. But the m e c h a n i s m by w h i c h Gn-RH b i n d s and regulates growth in CUME-1 cells remains to be determined. It has been reported that p53 gene mutations are the most frequent genetic changes in h u m a n cancers [32]. Imm u n o h i s t o c h e m i c a l studies on endometrial adenocarcin o m a tissues have s h o w n that overexpression of p53 has been found i n about 20% [33, 34]. Yaginuma a n d Westphal [35] also reported that sequencing analysis of the entire coding region revealed p53 mutations in all six endometrial carcinoma cell lines tested. On the contrary, we found that CUME-1 cell line contained no p53 mutation.

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This cell line may be useful i n studying in the vitro and in vivo properties of h u m a n endometrial adenocarcinoma. This work was supported by a grant from the Ministry of Science and Technology (95-I-1-093).

REFERENCES

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