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- Kotova, Natalia, 1975-, et al.
(författare)
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Flow cytometric determination of HPRT-variantsin human peripheral blood lymphocytes
- 2002
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Ingår i: Mutation research. - 0027-5107 .- 1873-135X. ; 499:1, s. 63-71
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Tidskriftsartikel (refereegranskat)abstract
- Hypoxanthine guanine phosphoribosyl transferase (HPRT) deficient human peripheral blood lymphocytes are usually enumerated either by the cloning assay or by the autoradiographic short-term assay. The short-term approach presented here is based on flow cytometric (FCM) scoring of 6-thioguanine (6-TG) resistant lymphocytes. HPRT-variants are enumerated on the basis of both DNA synthesis (by use of immunofluorescent detection of incorporated 5-bromo-2-deoxyuridine, BrdU) and total DNA content (by propidium iodide (PI) incorporation) of proliferating cells, i.e. the cells must both be labelled with BrdU and reside in late-S or G(2) phase in order to be scored as a HPRT-variant. This approach is combined with a stringent discrimination of false-positive events, minimising occurrence of phenocopies or other non-specifically labelled cells that might falsely be scored as true HPRT-variants. The HPRT-variant frequency (V-f) found by the presented method varied between 0.8 x 10(-5) and 5.8 x 10(-5) for healthy male and female donors aged between 20 and 74 years. There was no significant gender difference in V-f. A strong linear correlation was found between HPRT-variant frequency and age, showing an increase of 0.56 x 10(-6) per year of age (r(2) = 0.62, P < 0.001). The frequencies of false-positive events found showed a mean of 0.22 x 10(-5) in comparison with a pooled mean V-f of 2.87 x 10(-5). There was no significant age effect on the frequency of false events (r(2) = = 0.15, P < 0.095). The method presented here may provide a rapid and sensitive alternative to the autoradiographic technique for the short-term enumeration of HPRT-variants.
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| 2. |
- Li, Li, et al.
(författare)
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Induction of apoptosis or necrosis in human endometrial cancer cells by 2-Methoxyestradiol
- 2004
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Ingår i: Anticancer Research. - 0250-7005 .- 1791-7530. ; 24:6, s. 3983-3990
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:We investigated the effects of 2methoxyestradiol (2-ME), an endogenous estrogenic metabolite, on human endometrial cancer HEC-1-A and RL-95-2 cell lines.MATERIALS AND METHODS:After exposure of HEC-1-A and RL-95-2 cells to 2-ME, the morphological changes were evaluated by acridine orange staining and transmission electron microscopy. Cell cycle progress, apoptosis and necrosis were assessed by flow cytometry, DNA fragmentation and Western blot.RESULTS:2-ME inhibited cell growth by blocking the S- and G2/M-phase in both cell lines, by inducing apoptosis in HEC-1-A cells and by causing necrosis in RL-95-2 cells. Apoptosis, on HEC-1-A cells, was accompanied by an increased expression of iNOS and STAT1. This apoptotic effect was prevented by the iNOS inhibitor 1400W and eliminated by the caspase inhibitor Z-VAD-FMK. Necrosis, on RL-95-2 cells, was due to a severe disruption of the mitochondrial membrane potential. 2-ME had no significant effect on normal human endometrial cells.CONCLUSION:The data suggest that 2-ME has an antitumor effect on human endometrial carcinoma cells (HEC-1-A and RL-95-2) and may contribute as a new therapeutic agent for endometrial cancer patients.
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