Establishment and Characterization of Human Ovarian Carcinoma Cell Lines

doi:10.1006/gyno.1997.4785

Gynecologic Oncology

Volume 66, Issue 3, September 1997, Pages 378-387

https://doi.org/10.1006/gyno.1997.4785 Get rights and content

Abstract

Five human ovarian carcinoma cell lines cultured from primary and metastatic tumors of Korean patients were characterized. These lines were isolated from two papillary serous cystadenocarcinomas, two endometrioid carcinomas, and one malignant Brenner tumor. It was shown that the growth of these cell lines was stable when cultured after at least 20 passages. Population doubling times varied from 40 to 67 hr. All lines showed high viability and were proven by DNA fingerprinting analysis to be unique. Contamination by mycoplasma or bacteria was excluded. In two lines, SNU-8 and SNU-840, an elevated level of CA125 antigen secretion could be detected, whereas CEA was undetectable in all five lines. Four different mutations in functional and highly conserved regions of the p53 gene were identified in three of our five lines (60%), namely in SNU-119, SNU-251, and SNU-563. Included were two missense mutations, one in-frame 3-base-pair deletion, and one out-of-frame 1-base-pair deletion. It is interesting to note that one of these three lines, SNU-251, presented an additional simultaneous nonsense mutation of the BRCA1 gene and missense mutation of the hMLH1 gene. In its lacking both wild-type alleles of the BRCA1 gene, SNU-251 might serve as an unusual and importantin vitromodel for studies related to ovarian carcinoma and the BRCA1 gene. It is thus likely that the establishment and characterization of these permanent human ovarian carcinoma cell lines in continuous cultures can provide useful tools forin vitrostudies related to human ovarian carcinomas.

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Cited by (25)

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation
2013, Molecular Oncology
Citation Excerpt :
This discrepancy is likely due to many cell lines being classified as adenocarcinoma, rather than into epithelial subgroups. Interestingly, although the majority of BRCA1 mutations occur in serous ovarian cancer (Berchuck et al., 1998; Lakhani et al., 2004) the SNU-251 BRCA1 mutant is derived from an endometrial ovarian tumour (Yuan et al., 1997). The PEO1, PEO4 and PEO6 cell lines which have reversions of a BRCA2 mutation are of also of serous origin from the same patient (Langdon et al., 1988)
Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6–13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors.
The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines.
The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311.
The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.
DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination
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In this report, we profiled the most widely used endometrial and ovarian cell lines and discovered several examples of misidentification, redundancy and cross-contamination. Genotyping and HPV testing of ovarian cancer cell lines identified eight (BG-1 [64], CH1/CH1cisR [72], 222 [75], C13 [76], A2008 [77,78], OV2008, SKOV-4 and SKOV-6 [79]) as previously existing, breast cancer, teratocarcinoma or cervical cancer cell lines. In addition, two ‘normal ovarian’ cell lines, NOSE06 and NOSE07 [80], were genotyped as the ovarian cancer line DOV-13 [81].
Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines.
Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed.
Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively.
Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.
BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells
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The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERα signaling. However, many ERα-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERα signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERα-negative cells. We previously noticed that both ERα-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERα-negative cell lines even exceeded its over-expression level in ERα-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERα-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.
Establishment and characterization of xenografts and cancer cell cultures derived from BRCA1 -/- epithelial ovarian cancers
2006, European Journal of Cancer
The BRCA1 gene is responsible for a high number of hereditary breast and ovarian cancers that cluster in families with a strong genetic predisposition. Despite intense investigation, the accumulating findings on BRCA1 biological functions have not yet been translated into specific therapeutic approaches, also due to the lack of suitable experimental models. The purpose of this study was to establish and characterize cell cultures and xenografts from patients with BRCA1 −/− ovarian cancers. We derived two ovarian cancer cell lines, termed PD-OVCA1 and PD-OVCA2, both from patients previously treated with chemotherapy, that propagate in SCID mice as well as in vitro for a limited number of passages. Both cell lines expressed cytokeratins and the CA125 tumour marker. A detailed molecular characterization highlighted both constitutive and somatic genetic events that abrogate BRCA1 gene function. Both cell lines were shown to lose the wild type BRCA1 allele; intriguingly, these deletions were apparently accompanied by gain of one or more copies of the mutant alleles. Finally, a genomic profile of major chromosomal aberrations was obtained by the Multiplex Ligation-dependent Probe Amplification (MLPA) technique, which disclosed chromosomal imbalances targeting specific genes in each cell line. The PD-OVCA1 and PD-OVCA2 ovarian cancer cell lines will provide a valuable tool for new experimental models for the study of BRCA1-associated tumour biology.
Inhibition of human telomerase reverse transcriptase gene expression by BRCA1 in human ovarian cancer cells
2003, Biochemical and Biophysical Research Communications
Citation Excerpt :
The ovarian cancer cell line SNU-251 was a gift from Dr. Jae-Gahb Park (Korea Cell Line Bank, Seoul National University, Korea) and further characterized by Zhou et al. [24]. Briefly, the SNU-251 cell line is an endometrioid ovarian cell line that carries a single-base G-to-A transition in exon 23, resulting in a stop codon at amino acid 1815 of BRCA1 and a predicted loss of half of the second BRCA1 C-terminal (BRCT) repeat [25]. All cell lines were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) in the presence of antibiotics.
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, is responsible for the synthesis and maintenance of the telomeric repeats at the distal ends of human chromosomes. Telomerase expression is repressed in normal human cells and is activated in immortal cells and during tumorigenesis, but the mechanism by which telomerase expression is regulated is not fully understood. Previous studies have shown that c-Myc stimulates hTERT transcription through the binding sites located on the hTERT promoter. In this study, we sought to determine whether BRCA1 inhibits hTERT transcription through its direct interaction with c-Myc. In ovarian cancer cells, c-Myc increased hTERT expression by threefold and BRCA1 completely abrogated this activity. A mutation in the c-Myc-binding site (E-box) of the hTERT promoter resulted in the loss of activation by c-Myc and in the loss of inhibition by BRCA1. Deletion of the c-Myc-binding domain in BRCA1 resulted in the loss of BRCA1’s ability to inhibit transcription of the hTERT promoter. In addition, BRCA1 associates with c-Myc and inhibits the binding activity of c-Myc to the hTERT promoter. Our data indicate that BRCA1 is involved in regulating cellular immortalization through the modulation of c-Myc on the hTERT promoter.
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^☆: Supported (in part) by the Korea Science and Engineering Foundation (KOSEF-CRC-94K2-0402-04-00-3) through the Cancer Research Center at Seoul National University.

^☆☆: R, J, HayJ, G, ParkA, Gradar, eds.

²: To whom correspondence and reprint requests should be addressed at Laboratory of Cell Biology, Cancer Research Institute, Seoul National University College of Medicine, 28, Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Fax: 82-2-742-4727. E-mail: [email protected].

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Regular ArticleEstablishment and Characterization of Human Ovarian Carcinoma Cell Lines☆,☆☆

Abstract

Am J Obstet Gynecol

Cancer statistics

Ca Cancer J Clin

Gynecologic Oncology: Fundamental Principles and Clinical Practice

Immunopathologic common surfaces antigens of human ovarian tumors of serous endometrioid and clear cell types

Am J Clin Pathol

A new human ovarian carcinoma cell line establishment and analysis of tumor associated markers

Oncology

Establishment and characterization of an estrogen producing human ovarian granulosa tumor cell line

J Natl Cancer Inst

Establishment and characterization of three new human ovarian carcinoma cell lines and initial evaluation of their potential in experimental chemotherapy studies

Int J Cancer

Establishment and characterization of four human epithelial ovarian carcinoma cell lines

Cancer

Reactivity of a monoclonal antibody with human ovarian carcinoma

J Clin Invest

Absence of HeLa cell contamination in 169 cell lines derived from human tumors

J Natl Cancer Inst

Sensitivity to taxid derivatives of a newly established human endometrioid ovarian adenocarcinoma radioresistant cell line

Anticancer Res

Catalogue of Cell Lines and Hybridomas

Growth of cell lines and clinical specimens of human non-small-cell lung cancer in a serum-free defined medium

Cancer Res

Characteristics of cell lines established from human colorectal carcinoma

Cancer Res

Culture of Animal Cells

Detection of Mycoplasma Contamination

Monoclonal Antibodies: Principles and Practice

Molecular Cloning: A Laboratory Manual

Regular Article
Establishment and Characterization of Human Ovarian Carcinoma Cell Lines☆,☆☆