Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors

O’Brien, Melissa C.; Healy, Stephen F.; Raney, Shula; Hurst, Josephine M.; Avner, Barry P.; Hanly, Andrew; Mies, Carolyn; Freeman, James W.; Snow, Christopher; Koester, Steven K.; Bolton, Wade E.

doi:10.1002/(sici)1097-0320(19970501)28:1<81::aid-cyto10>3.0.co;2-n

Cited by 48 publications

(22 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found an approximately 8% difference in the dead cell ratio between the trauma group and the control group. As the Live/Dead stain utilizes the substrate calcein for intracellular esterases and an ethidium dimer which penetrates disrupted cell membranes and intercalates with the DNA in the cell nucleus, this method detects early necrotic rather than apoptotic cell death [39,42]. In a recent study by Whiteside et al the effect of impact loading on the viability of articular cartilage was investigated using confocal microscopy.…”

Section: Discussionmentioning

confidence: 99%

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

et al. 2007

View full text Add to dashboard Cite

There is a major controversy whether spinal trauma with vertebral endplate fractures can result in posttraumatic disc degeneration. Intervertebral discs, which are adjacent to burst endplates, are frequently removed and an intercorporal spondylodesis is performed. In any case, the biological effects within the discs following endplate factures are poorly elucidated to date. The aim of our investigations was therefore to establish a novel disc/endplate trauma culture model to reproducibly induce endplate fractures and investigate concurrent disc changes in vitro. This model is based on a full-organ disc/endplate culture system, which has been validated by the authors before. Intervertebral disc/endplate specimens were isolated from Burgundy rabbits and cultured in standard media (DMEM/ F12, 10%FCS). Burst endplate fractures were induced in half of the specimens with a custom-made fracture device and subsequently cultured for 9 days. The biological effects such as necrotic or apoptotic cell death and the expression of pro-apoptotic genes and other genes involved in organ degeneration, e.g. matrix metalloproteinases (MMPs) were analyzed. Cell damage was assessed by quantification of the lactate dehydrogenase (LDH) activity in the supernatant. The expression of genes involved in the cellular apoptotic pathway (caspase 3) and the pro-apoptotic proteins FasL and TNF-a were monitored. The results demonstrate that LDH levels increased significantly post trauma compared to the control and remained elevated for 3 days. Furthermore, a constant up-regulation of the caspase 3 gene in both disc compartments was present. The pro-apoptotic proteins FasL and TNF-a were up regulated predominantly in the nucleus whereas the MMP-1 and -13 transcripts (collagenases) were increased in both disc structures. From this study we can conclude that endplate burst fractures result in both necrotic and apoptotic cell death in nucleus and annulus tissue. Moreover, FasL and TNF-a expression by nucleus cells may lead to continued apoptosis induced by Fas-and TNF-a receptor bearing cells. In addition TNF-a over-expression has potentially deleterious effects on disc metabolism such as overexpression of matrix proteinases. Taken together, the short term biological response of the disc following endplate fracture exhibits characteristics, which may initiate the degeneration of the organ.

show abstract

Section: Discussionmentioning

confidence: 99%

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

et al. 2007

View full text Add to dashboard Cite

show abstract

“…We observed a wider standard deviation and some spurious high values by flow cytometry compared with corresponding image analysis. Flow cytometric studies measuring apoptosis using either DNA conformational changes (20) or DNA fragmentation by sub-G 1 peak quantitation (12) have reported medians of 5.5% and 11.5% on 16 and 10 breast tumors, respectively. Previous TUNEL assay studies of solid tumors using flow or laser scanning cytometry have shown widely variable values of apoptosis, ranging from 2.1% to 27% (13)(14)(15)21).…”

Section: Discussionmentioning

confidence: 99%

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma

et al. 2001

View full text Add to dashboard Cite

The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3 end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. Key terms: TUNEL assay; apoptosis; breast carcinoma; flow cytometry; image analysis Programmed cell death (apoptosis) results from a cascade of events in response to a variety of stimuli (1,2). This vital process plays an important role in normal cellular homeostasis, tumor cell dynamics, and response to anticancer therapies (3,4). The assessment of apoptosis is therefore of interest in the biological evaluation of tumor kinetics and in the potential prediction of clinical response to treatment.Several techniques to detect different events in the temporal sequence of this process have been developed and used to quantitate apoptotic cells (1,2,5). Methods to measure early changes in cell membrane permeability, phospholipid asymmetry, mitochondrial membrane potential, and caspase activation have been used to identify apoptotic cells in experimental systems. In contrast, studies of apoptosis in solid tumors have mainly utilized techniques that measure later events such as DNA fragmentation. Of these, the 3Ј end-labeling technique (TUNEL assay) appears to be the method of choice for many studies (6 -15).The majority of breast carcinoma studies have been histology-based TUNEL assays that use immunoperoxidase methods. These techniques are less sensitive than the corresponding fluorescence-based assays which, in addition, allow multiparametric analysis of DNA fragmentation with the quantitation of DNA content and cellular antigens or fluorescence in situ hybridization (FISH) using image analysis, laser scanning, or flow cytometry.In this study, we applied the fluorescent TUNEL assay to fine needle samples of breast carcinomas and compared the results obtained by flow cytometry and image analysis. MATERIALS AND METHODSThirty-one fine needle samples from 30 primary breast carcinoma patients were prospectively acquired at the Institut Curie and formed the material for this study. Specimens were placed immediately into 1 ml RPMI 1640 (Sigma, St. Louis, MO) with 10% fetal calf serum (FCS;

show abstract

“…All cultures were subconfluent at the time of collection. Cultures were collected for fixation, stained with propidium iodide, and analyzed using flow cytometry as previously described by the Clinical Services Program at National Cancer Institute-Frederick (28). To evaluate the activation of the G 2 cell cycle checkpoint, mitotic cells were distinguished from G 2 cells and the mitotic index was determined according to the expression of phosphorylated histone H3 (Upstate Biotechnology, Charlottesville, VA) as detected in the 4N DNA content population using the flow cytometric method of Xu et al (29).…”

Section: Methodsmentioning

confidence: 99%

Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine

Dote

Cerna

Burgan

et al. 2005

Clinical Cancer Research

View full text Add to dashboard Cite

Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and14-3-3r) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, gH2AX expression was determined.Whereas zebularine had no effect on radiation-induced gH2AX foci at 1hour, the number of gH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251xenografts; irradiation of U251tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay.These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.

show abstract

Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors

Cited by 48 publications

References 36 publications

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma

Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine

Contact Info

Product

Resources

About