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Sumiko Watanabe, Tsunehisa Kaku, Maki Kusaba, Natsuki Eguchi, Kazunori Nishimura, Setsuko Murata, Setsuo Sugishima, Tsuyoshi Iwasaka, Formation Mechanism of Binucleated HeLa Cells, 38th European Congress of Cytology 2014, 2014.09, Introduction:
Many cytological studies have reported an increase in the numbers of binucleated cells in various tumors. We found that the numbers of binucleated HeLa cells in a serum-free condition was significantly higher than that in a conventional condition. Two reports have revealed different mechanisms for binucleated cell formation: ‘cell–cell fusion’ and ‘abnormal cytokinesis’. Here we attempted to elucidate the formation mechanism of binucleated HeLa cells using CellTracker™.
Materials and Methods:
We grew HeLa cells on chamber slides for 4 days in DMEM supplemented with fetal bovine serum (10% medium). After that, we removed the 10% medium and cultured HeLa cells in DMEM without fetal bovine serum (0% medium) including CellTracker™ for 45 minutes. Then co-culture 2 kinds cells treated with the two different CellTracker™ probes (Red or Green) in 0% medium for 3 days. We observed the cytoplasmic staining condition of binecleated HeLa cells by confocal laser scanning microscopy (CLSM).
Results:
We observed 54 binucleated HeLa cells. The ratio of cytoplasmic stained with red, green and yellow (means stained with both) was 40.7%, 57.4% and 1.9%, respectively. The ratio of single-color binucleated cells was 98.1%. If the formation mechanism is cell–cell fusion, cytoplasms of binucleated cells might be stained with red, green and yellow and each theoretical frequency of binucleated cells was about 25%, 25% and 50%, respectively. There were large discrepancies in experimental and theoretical values of cell–cell fusion.
Conclusion:
Binucleated HeLa cells might result from abnormal cytokinesis.
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渡邊 壽美子, 加来 恒壽, 大喜 雅文, 田宮 貞史, 杉島 節夫, 村田節子, 大石 善丈, 横山正俊, 柏村正道, 柏村賀子, 岩坂剛, Correlation between nuclear chromatin pattern and cell cycle, 18th International Congress Of Cytology, 2013.05, Objectives:
The chromatin pattern of nuclei is one of the most important criteria for cytological diagnosis. However many terms like fine granular, coarse granular, opaque and so on, very few reports have actually referred to the need to clarify the definition of chromatin pattern. In the present study, we quantitatively analyzed nuclear chromatin distribution in 3 cancer cell lines and examined the relationship between the chromatin distribution pattern and the cell cycle.
Materials and Methods:
We used three cultured cell lines; HeLa (moderate differentiated cervical adenocarcinoma), TMCC-1 (undifferentiated cervical adenicarcinoma) and T-24 (high grade urothelial carcinoma). Cells were cultured in Dulbecco’s modified Eagle’s medium: DMEM supplemented with 10% fetal calf serum and 1x antibiotic-antimycotic solution at 37oC in 5 % CO2 for 3,5,7 and 10 days on chamber slides. Three hundreds cells were selected at random in each slide. Cell images stained with the Papanicolaou method were acquired by a digital camera attached to a microscope and saved in a computer in JPEG format. Chromatin patterns were divided into 4 types; fine granular (F), Medium granular (M), coarse granular (C) and opaque (O). DNA contents were measured for the cell cycle analysis with flowcytometer.
Results:
Chromatin pattern compositions of the following days were similar between 3 cells. The ratio of C type was increasing (average of 3 cells; 1.7%, 3.0%, 11.0% and 10.3% was day 3, day 5, day 7 and day 10, respectively), while F type was decreasing (average of 3 cells; 69.0% 56.0%, 26.0% and 6.7% was day 3, day 5, day 7 and day 10, respectively). O type was appeared just only day 10. The ratio of G1 of 3 cells was increasing (average of 3 cells; 54.5% 68.1%, 72.7% and 74.5% was day 3, day 5, day 7 and day 10, respectively).
Conclusion:
It is suggested that there is a significant correlation between the nuclear chromatin pattern and the ratio of G1 phase of cell cycle.
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