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Early detection of cytogenetic damage from radiation or chemical agents by flow-cytometric analysis of micronucleated cd71-positive reticulocytes in human peripheral blood
S366
I. J. Radiation Oncology
● Biology ● Physics
Volume 60, Number 1, Supplement, 2004
aberrations and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Materials/Methods: Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. Cell death was induced by staurosporine (STS) and determined by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. Results: We found that M059K cells were much more sensitive to STS treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak and Fas. The cell death stimuli increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Conclusions: Our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.
2049
Early Detection of Cytogenetic Damage from Radiation or Chemical Agents by Flow-Cytometric Analysis of Micronucleated CD71-Positive Reticulocytes in Human Peripheral Blood
Y. Chen,1 S. D. Dertinger,2 K. Pandya,3 D. K. Torous,2 K. J. Brewer,4 T. Smudzin,1 R. K. Miller4 Radiation Oncology, University of Rochester, Rochester, NY, 2Litron Laboratories, Rochester, NY, 3Hematology/Oncology, University of Rochester, Rochester, NY, 4Obstetrics and Gynecology, University of Rochester, Rochester, NY 1
Purpose/Objective: Micronuclei (MN) are formed upon cell division in cells with DNA double-stand break(s) or dysfunctional mitotic spindle apparatus. Based on the detailed understanding of MN origin, the rodent-based micronucleus test has become the most widely utilized in vivo system for evaluating chemicals’ clastogenic and aneugenic potential. The rodent-based tests are most typically performed as reticulocyte-(RET) based assays. Target cells for RET-based micronucleus assays were traditionally obtained from the bone marrow compartment. The application of this endpoint to peripheral blood has been limited due to the expected low sensitivity as a result of the effective splenic clearance of MN-containing reticulocytes (MN-RET) from circulation. Litron Laboratories has developed a flow cytometry-based method for quantifying the incidence of MN-RET in peripheral blood compartment of humans. This high-throughput scoring system utilizes a nucleic acid dye in combination with an erythrocyte maturity marker (fluorescent antibody against CD71). Together, these reagents allow for quantification of MN in the most immature fraction of RETs (MN-RETCD71⫹), thereby helping to reduce the effect of splenic filtration. This technology is capable of high rates of analysis and may offer rapid, efficient, and quantitative assessment of cytogenetic damages in humans by radiation or chemoradiation. We report a pilot study applying this methodology to the early detection of genotoxicity from radiation or chemotherapeutic agents using human peripheral blood samples. Materials/Methods: Eighteen patients receiving either radiation (n ⫽ 9), full-dose chemotherapy (n ⫽ 4), or combination chemoradiation (n ⫽ 5) for cancer treatments consented to an IRB-approved clinical protocol, which allows up to five daily blood sampling for the assay. Two ml of peripheral blood was drawn before treatment, and daily after treatment at approximately 24-hour intervals over the first course of the first week of therapy. Blood samples were fixed, and processed, stored, and stained according to the standard procedures developed by Litron Laboratories. The frequency of MN-RETCD71⫹ was measured using a single-laser flow cytometer providing 488nm excitation. Results: The majority of cancer patients receiving radiotherapy or chemotherapy demonstrated increased MN-RETCD71⫹ frequencies over the course of therapy. The increase was observed at as early as 24 hours after treatments, with the peak occurring between day 2 and day 4 after radiation or chemotherapy. The peak fold increases of MN-RETCD71⫹ ranged from 1.3 to 10.0 fold for radiation only, from 1.7 to 16.7 fold for chemoradiation, and from 10.7 to 26.7 fold for full-dose chemotherapy. There was an exponential increase of MN-RETCD71⫹ as a function of bone marrow suppression, which was measured by the percent reduction of peripheral RETs. There was also a positive correlation in MN-RETCD71⫹ increase with the volume of irradiated bone. Conclusions: This study demonstrates the feasibility of high-throughput flow cytometric analysis of MN-RETCD71⫹ in detecting cytogenetic damage by ionizing radiation and chemotherapeutic agents as early as 24 hours after exposure. The method is quantitative, sensitive, rapid, and is feasible with only 2 ml of human peripheral blood samples. This technology offers potential in the management of genotoxic exposure in the early detection, assessment of severity, monitoring status, and gauging therapeutic interventions. The work was supported by a NIEHS grant to Litron Laboratories (S.D.D.), number R44 ES010752-02.
2050
Cyclooxygenase-2 Inhibitor, Nimesulide, Improves Radiation Treatment Against Non-Small Cell Lung Cancer Both In Vitro and In Vivo
K. R. Grimes,1 G. W. Warren,2 D. K. St. Clair,3,1 W. H. St. Clair4,1 Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, 2College of Medicine, University of Kentucky, Lexington, KY, 3Graduate Center of Toxicology, University of Kentucky, Lexington, KY, 4Radiation Medicine, University of Kentucky, Lexington, KY 1
Purpose/Objective: Lung cancer is the leading cause of cancer related deaths in the United States. Despite improvements in radiation, surgery, and chemotherapy the 5 year survival statistics of non-small cell lung cancer (NSCLC) have improved little over the past two decades. It has been proposed that NF-B is a participant in the cytoprotection against several redox-mediated
I. J. Radiation Oncology
● Biology ● Physics
Volume 60, Number 1, Supplement, 2004
aberrations and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Materials/Methods: Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. Cell death was induced by staurosporine (STS) and determined by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. Results: We found that M059K cells were much more sensitive to STS treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak and Fas. The cell death stimuli increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Conclusions: Our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.
2049
Early Detection of Cytogenetic Damage from Radiation or Chemical Agents by Flow-Cytometric Analysis of Micronucleated CD71-Positive Reticulocytes in Human Peripheral Blood
Y. Chen,1 S. D. Dertinger,2 K. Pandya,3 D. K. Torous,2 K. J. Brewer,4 T. Smudzin,1 R. K. Miller4 Radiation Oncology, University of Rochester, Rochester, NY, 2Litron Laboratories, Rochester, NY, 3Hematology/Oncology, University of Rochester, Rochester, NY, 4Obstetrics and Gynecology, University of Rochester, Rochester, NY 1
Purpose/Objective: Micronuclei (MN) are formed upon cell division in cells with DNA double-stand break(s) or dysfunctional mitotic spindle apparatus. Based on the detailed understanding of MN origin, the rodent-based micronucleus test has become the most widely utilized in vivo system for evaluating chemicals’ clastogenic and aneugenic potential. The rodent-based tests are most typically performed as reticulocyte-(RET) based assays. Target cells for RET-based micronucleus assays were traditionally obtained from the bone marrow compartment. The application of this endpoint to peripheral blood has been limited due to the expected low sensitivity as a result of the effective splenic clearance of MN-containing reticulocytes (MN-RET) from circulation. Litron Laboratories has developed a flow cytometry-based method for quantifying the incidence of MN-RET in peripheral blood compartment of humans. This high-throughput scoring system utilizes a nucleic acid dye in combination with an erythrocyte maturity marker (fluorescent antibody against CD71). Together, these reagents allow for quantification of MN in the most immature fraction of RETs (MN-RETCD71⫹), thereby helping to reduce the effect of splenic filtration. This technology is capable of high rates of analysis and may offer rapid, efficient, and quantitative assessment of cytogenetic damages in humans by radiation or chemoradiation. We report a pilot study applying this methodology to the early detection of genotoxicity from radiation or chemotherapeutic agents using human peripheral blood samples. Materials/Methods: Eighteen patients receiving either radiation (n ⫽ 9), full-dose chemotherapy (n ⫽ 4), or combination chemoradiation (n ⫽ 5) for cancer treatments consented to an IRB-approved clinical protocol, which allows up to five daily blood sampling for the assay. Two ml of peripheral blood was drawn before treatment, and daily after treatment at approximately 24-hour intervals over the first course of the first week of therapy. Blood samples were fixed, and processed, stored, and stained according to the standard procedures developed by Litron Laboratories. The frequency of MN-RETCD71⫹ was measured using a single-laser flow cytometer providing 488nm excitation. Results: The majority of cancer patients receiving radiotherapy or chemotherapy demonstrated increased MN-RETCD71⫹ frequencies over the course of therapy. The increase was observed at as early as 24 hours after treatments, with the peak occurring between day 2 and day 4 after radiation or chemotherapy. The peak fold increases of MN-RETCD71⫹ ranged from 1.3 to 10.0 fold for radiation only, from 1.7 to 16.7 fold for chemoradiation, and from 10.7 to 26.7 fold for full-dose chemotherapy. There was an exponential increase of MN-RETCD71⫹ as a function of bone marrow suppression, which was measured by the percent reduction of peripheral RETs. There was also a positive correlation in MN-RETCD71⫹ increase with the volume of irradiated bone. Conclusions: This study demonstrates the feasibility of high-throughput flow cytometric analysis of MN-RETCD71⫹ in detecting cytogenetic damage by ionizing radiation and chemotherapeutic agents as early as 24 hours after exposure. The method is quantitative, sensitive, rapid, and is feasible with only 2 ml of human peripheral blood samples. This technology offers potential in the management of genotoxic exposure in the early detection, assessment of severity, monitoring status, and gauging therapeutic interventions. The work was supported by a NIEHS grant to Litron Laboratories (S.D.D.), number R44 ES010752-02.
2050
Cyclooxygenase-2 Inhibitor, Nimesulide, Improves Radiation Treatment Against Non-Small Cell Lung Cancer Both In Vitro and In Vivo
K. R. Grimes,1 G. W. Warren,2 D. K. St. Clair,3,1 W. H. St. Clair4,1 Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, 2College of Medicine, University of Kentucky, Lexington, KY, 3Graduate Center of Toxicology, University of Kentucky, Lexington, KY, 4Radiation Medicine, University of Kentucky, Lexington, KY 1
Purpose/Objective: Lung cancer is the leading cause of cancer related deaths in the United States. Despite improvements in radiation, surgery, and chemotherapy the 5 year survival statistics of non-small cell lung cancer (NSCLC) have improved little over the past two decades. It has been proposed that NF-B is a participant in the cytoprotection against several redox-mediated