The objective of this study was to study the human ovarian
September 26, 2017
The objective of this study was to study the human ovarian cancer cell line CABA I by means of short tandem repeats (STR) profiling and cytogenetic analysis in order to prevent future misidentification or cross-contamination and verify its stability during cultivation. that this STR profile is usually reliable and could be used for the regular authentication of CABA I over time. It should be emphasized, however, that of the 16 loci generally used in human STR profiles, only 3 were properly detectable in CABA I. The data highlight that this CABA I cell collection demonstrates an anomalous STR profile that does not fully change the criteria currently utilized for the identification of human cells; in spite of this, it remains stable during the maintainance. Moreover, the genetic instability of the CABA I cell collection overlaps with those observed in tumor cells, making it a suitable candidate to analyze, cell collection research; specifically, ovarian malignancy studies may use several human cell lines such as OVCAR3 (5), SK-OV-3 (6), A2780 (7), IGROV1 (8) or OAW42 (9). CABA I is an ovarian malignancy cell line of epithelial origin, which was established from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. CABA I cell growth is anchorage dependent and very quick (the doubling time is approximately 18 h); preliminary cytogenetic analysis indicated a modal chromosome quantity of 57C58, with 44 clonal structural aberrations and only few chromosomes appearing morphologically normal (10). It has previously been exhibited that growth and phenotypic characteristics are managed both in early and late passages, suggesting that this CABA I cell collection provides a suitable model system in order to investigate the cellular and molecular events involved in ovarian carcinogenesis (10). Thus, since then, many studies have concentrated on CABA I behavior in malignancy progression, with particular interest being shown in relation to the release of extracellular vesicles (11C18). For some years, the short tandem repeats (STR) profiling has been the international research standard for the identification of cell lines (19C22), and thus in the present study we proceeded to subject CABA I to this analysis to prevent future 56392-17-7 IC50 misidentification or cross-contaminations during cultivation. Furthermore, the cell collection was analyzed by classical and molecular CPB2 cytogenetic techniques: we selected two different passages, the 18th and 38th, to identify chromosomal aberrations and the karyotypic development of this cell collection. Materials and methods CABA I cells The 18th and 38th passages of CABA I cells were produced as monolayers in RPMI-1640 with 5% fetal calf serum, 2 mM glutamine and penicillin 100 U/ml (all materials are 56392-17-7 IC50 from Euroclone, Devon, UK). Cells at passages 18 and 38 were tested for mycoplasma contamination and the result was unfavorable. Cells exceeded from 18th to 38th passages in approximately 15 weeks. Cytogenetic analyses Standard cytogenetic techniques (23) were used on CABA I cells at the 18th and 38th passages in order to identify chromosomal aberrations and the karyotypic development of this cell collection. In addition, every metaphase was analyzed by sequential GTG-banding and fluorescence in situ hybridization (FISH) with whole chromosome painting probes specific for each chromosome. Briefly, metaphases stained with giemsa answer after partial trypsin digestion (GTG-banding) were observed under a light field microscope (Leica Aristoplan microscope; Leica, Wetzlar, Germany), captured with PSI MacKtype software and finally destained three times in methanol. The slides were then washed in 2X SSC answer, heated at 70C in SSC/formamide treatment for denature target chromosome DNA, and hybridized with FISH probes specific to a whole chromosome. Observation under a fluorescence microscope allowed us to capture again the same GTG-banded metaphases previously observed and analyze the hybridization on markers chromosomes of the CABA I cell collection. DNA extraction The DNA contained in CABA I cells, at the 18th and 38th passages, was extracted from approximately 5106 cells using an automatic extractor MagNA Pure Compact system (Roche Diagnostics, Basel, Switzerland). The procedure involves several steps, consisting of preparatory cell disruption and protein digestion caused by the addition of lysis buffer and proteinase K, the formation of nucleic acid-bead complexes caused by nucleic acid binding to the surface of magnetic glass particles and subsequent magnetic separation; after washing to remove cell debris, nucleic acid is usually eluted at high temperatures with simultaneous removal of the magnetic glass particles. Thus, 56392-17-7 IC50 44.4 and 136 ng/long term culture effects around the genetic features of the CABA I cell collection, these examinations were repeated on cells at the 38th passage. It was not possible to recover the donor’s initial tissue, and initial passage stocks of CABA I cells are no longer.