Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry

Grabarek, Jerzy; Ardelt, Barbara; Kunicki, Jan; Darzynkiewicz, Zbigniew

doi:10.1002/cyto.10150

Cited by 26 publications

(32 citation statements)

References 24 publications

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“…5A). Resistance of apoptotic cells to the nonionic detergent Triton X-100 was reported to correlate with activity of TG2, which mediates protein cross-linking (39). During apoptosis, the proportion of 7-AAD + cells and the level of 3B9 binding to these cells were comparable irrespective of Triton X-100 treatment (Fig.…”

Section: Resultsmentioning

confidence: 89%

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The La Autoantigen Is a Malignancy-Associated Cell Death Target That Is Induced by DNA-Damaging Drugs

Al-Ejeh

Darby²,

Brown³

2007

Clinical Cancer Research

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Purpose: To evaluate the La autoantigen as a target for specific monoclonal antibody (mAb) binding in dead cancer cells after use of DNA-damaging chemotherapy. Experimental Design: In vitro studies of La-specific 3B9 mAb binding to malignant and normal primary cells with and without cytotoxic drug treatment were done using immunoblotting and flow cytometry. Chromatin-binding studies and immunofluorescence detection of gH2AX as a marker of DNA double-stranded breaks together with 3B9 binding assays were done to measure DNA damage responses. Incorporation of a transglutaminase 2 (TG2) substrate and TG2 inhibition were studied to measure protein cross-linking in dead cells. Results: La was overexpressed in human cancer cell lines with respect to normal primary cells. Within 3 h of the DNA-damaging stimulus, La became chromatin bound when it colocalized with gH2AX. Later, after the stimulus produced cell death, La-specific 3B9 mAb bound specifically and preferentially in the cytoplasm of dead cancer cells. Moreover, 3B9 binding to dead cancer cells increased with increasing DNA damage. Both La and 3B9 became cross-linked in dead cancer cells viaTG2 activity. Conclusion: La autoantigen represents a promising cancer cell death target to determine chemotherapy response because its expression was selectively induced in dead cancer cells after DNAdamaging chemotherapy.

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Section: Resultsmentioning

confidence: 89%

“…TG2-mediated protein-protein cross-linking in apoptotic cells was investigated using a modification of a previously described method (39). Briefly, cells were resuspended at 1 Â 10 6 /mL in PBS containing 1% Triton X-100, 0.2 Ag/mL sulforhodamine-101, and 0.1 Amol/L SytoxGreen.…”

Section: Methodsmentioning

confidence: 99%

The La Autoantigen Is a Malignancy-Associated Cell Death Target That Is Induced by DNA-Damaging Drugs

Al-Ejeh

Darby²,

Brown³

2007

Clinical Cancer Research

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“…Moreover, 3B9 still bound EL4 cells killed with cytotoxic drugs after treatment with nonionic detergent, which suggested that La was cross-linked in dead cells (ref. 37; Fig. 1D).…”

Section: Resultsmentioning

confidence: 91%

In vivo Targeting of Dead Tumor Cells in a Murine Tumor Model Using a Monoclonal Antibody Specific for the La Autoantigen

Al-Ejeh

Darby

Pensa

et al. 2007

Clinical Cancer Research

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Purpose: To investigate the potential of the La-specific monoclonal antibody (mAb) 3B9 as an in vivo tumor-targeting agent. Experimental Design: The murine EL4 lymphoma cell line was used for in vitro studies and the EL4 model in which apoptosis was induced with cyclophosphamide and etoposide was used for in vivo studies. In vitro studies compared 3B9 binding in the EL4 cell with that in its counterpart primary cell type of the thymocyte. For in vivo studies, 3B9 was intrinsically or extrinsically labeled with carbon-14 or 1,4,7,10-tetra-azacylododecane-N,N ¶,N 00 ,N 0000 -tetraacetic acidî ndium-111, respectively, and biodistribution of the radiotracers was investigated in EL4 tumorbearing mice, which were treated or not with chemotherapy. Results: La-specific 3B9 mAb bound EL4 cells rather than thymocytes, and binding was detergent resistant. 3B9 binding to dead EL4 cells in vitro was specific, rapid, and saturable. Significantly, more 3B9 bound dead EL4 tumor explant cells after host mice were treated with chemotherapy, which suggested that DNA damage induced 3B9 binding. Tumor binding of 3B9 in vivo was antigen specific and increased significantly after chemotherapy. Tumor accumulation of 3B9 peaked at f50% of the injected dose per gram of tumor 72 h after chemotherapy and correlated with increased tumor cell death. Tumor/organ ratios of 3B9 biodistribution, which included the tumor/blood ratio, exceeded unity 48 or more hours after chemotherapy. Conclusions: La-specific mAb selectively targeted dead tumor cells in vivo, and targeting was augmented by cytotoxic chemotherapy. This novel cell death radioligand may be useful both for radioimmunoscintigraphy and radioimmunotherapy.

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“…Moreover, this cytometer is more versatile and have been used in several scientific studies (35,40,41).…”

Section: Discussionmentioning

confidence: 99%

Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water

et al. 2007

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Background: Cryptosporidium detection in water and environmental samples has increased during the last years, largely due to an increase in the number of reported waterborne outbreaks of cryptosporidiosis and the implementation of new regulations about Cryptosporidium monitoring in water supplies. The aim of this study was to validate and compare the capacity of two laser scanning cytometers commercially available (LSC® and ChemScanRDI®), against manual microscopic enumeration of Cryptosporidium oocysts in surface water and reference material samples. Methods: Reference material and surface water samples were analysed by two laser scanning cytometers methodologies and by manual epifluorescence microscopy. Two mAbs from commercial suppliers were used to evaluate background reduction. Results: Highly significant correlations were obtain between both cytometers (R2 = 0.99) and with manual microscopy (R2 = 0.98), showing that oocysts counts made by cytometers were equivalent to those obtained with conventional methods. We observed a variability in oocysts counts when different antibodies where used with laser scanning cytometers and manual microscopy. Conclusions: This study showed the efficacy of the laser scanning technology (LSC® and ChemScanRDI®), as an automated and a more standardized alternative to manual epifluorescence microscopy examination, for Cryptosporidium detection in water samples. High quality antibodies are needed for automated enumeration as well as for manual microscope observations. © 2007 International Society for Analytical Cytology

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Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry

Cited by 26 publications

References 24 publications

The La Autoantigen Is a Malignancy-Associated Cell Death Target That Is Induced by DNA-Damaging Drugs

The La Autoantigen Is a Malignancy-Associated Cell Death Target That Is Induced by DNA-Damaging Drugs

In vivo Targeting of Dead Tumor Cells in a Murine Tumor Model Using a Monoclonal Antibody Specific for the La Autoantigen

Comparative study between two laser scanning cytometers and epifluorescence microscopy for the detection of Cryptosporidium oocysts in water

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