Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes

1994 ◽  
Vol 72 (11-12) ◽  
pp. 475-482 ◽  
Author(s):  
S. P. Cregan ◽  
D. R. Boreham ◽  
P. R. Walker ◽  
D. L. Brown ◽  
R. E. J. Mitchel
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Adaptive Response ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Fluorescence Analysis ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Protective Mechanism ◽  
Radiation Induced ◽  
Induced Apoptosis

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3′-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose γ-radiation (0.1 Gy) or mild hyperthermia (40 °C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.Key words: apoptosis, adaptive response, ionizing radiation, hyperthermia.

scholarly journals DNA damage response and preleukemic fusion genes induced by ionizing radiation in umbilical cord blood hematopoietic stem cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pavol Kosik ◽  
Matus Durdik ◽  
Lukas Jakl ◽  
Milan Skorvaga ◽  
Eva Markova ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Fusion Genes ◽  
Hematopoietic Stem ◽  
Radiation Induced ◽  
Induced Apoptosis

Abstract There is clear evidence that ionizing radiation (IR) causes leukemia. For many types of leukemia, the preleukemic fusion genes (PFG), as consequences of DNA damage and chromosomal translocations, occur in hematopoietic stem and progenitor cells (HSPC) in utero and could be detected in umbilical cord blood (UCB) of newborns. However, relatively limited information is available about radiation-induced apoptosis, DNA damage and PFG formation in human HSPC. In this study we revealed that CD34+ HSPC compared to lymphocytes: (i) are extremely radio-resistant showing delayed time kinetics of apoptosis, (ii) accumulate lower level of endogenous DNA damage/early apoptotic γH2AX pan-stained cells, (iii) have higher level of radiation-induced 53BP1 and γH2AX/53BP1 co-localized DNA double stranded breaks, and (iv) after low dose of IR may form very low level of BCR-ABL PFG. Within CD34+ HSPC we identified CD34+CD38+ progenitor cells as a highly apoptosis-resistant population, while CD34+CD38− hematopoietic stem/multipotent progenitor cells (HSC/MPP) as a population very sensitive to radiation-induced apoptosis. Our study provides critical insights into how human HSPC respond to IR in the context of DNA damage, apoptosis and PFG.

scholarly journals Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3843
Author(s):  
Jeffrey R. Whiteaker ◽  
Tao Wang ◽  
Lei Zhao ◽  
Regine M. Schoenherr ◽  
Jacob J. Kennedy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Limit Of Quantification ◽  
Peripheral Blood Mononuclear ◽  
Atm Kinase ◽  
Support Mechanism ◽  
Radiation Induced ◽  
Pharmacodynamic Biomarkers ◽  
Preclinical And Clinical Studies

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Granulocyte-Macrophage Colony-Stimulating Factor Rescues TF-1 Leukemia Cells From Ionizing Radiation-Induced Apoptosis Through a Pathway Mediated by Protein Kinase Cα

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 416-424 ◽  
Author(s):  
Mary L. Kelly ◽  
Yan Tang ◽  
Nitsa Rosensweig ◽  
Sanda Clejan ◽  
Barbara S. Beckman
Keyword(s):  
Protein Kinase ◽  
Ionizing Radiation ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Pkc Isoforms ◽  
Radiation Induced ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Induced Apoptosis ◽  
Stimulating Factor

Abstract Protein kinase C (PKC) activity has a recognized role in mediating apoptosis. However, the role of individual PKC isoforms in apoptosis is poorly defined. Therefore, we investigated the translocation of individual PKC isoforms during radiation-induced apoptosis with and without rescue from apoptosis by granulocyte-macrophage colony-stimulating factor (GM-CSF) in the human erythroleukemia cell line TF-1. PKCα was translocated from the particulate to cytosolic fraction of TF-1 cells within 5 minutes of treatment with apoptosis-inducing levels of ionizing radiation. However, this postirradiation translocation did not occur when cells were rescued from apoptosis by GM-CSF. Furthermore, treatment of cells with Gö6976, an inhibitor of classical PKC isoforms, abrogated the rescue effect of GM-CSF. The calcium-independent novel PKC isoform, PKCδ appeared to be degraded in both the particulate and cytosolic fractions of TF-1 cells after treatment with apoptosis-inducing levels of ionizing radiation in either the presence or absence of GM-CSF rescue. Levels of ceramide, a lipid mediator of apoptosis, were measured at 2, 4, 8, 10, and 60 minutes after treatment with ionizing radiation and were substantially reduced in TF-1 cells rescued from apoptosis by GM-CSF compared with apoptotic TF-1 cells. The largest decrease in ceramide production seen was at 4 minutes postirradiation, with a 46% reduction in ceramide levels in TF-1 cells rescued from apoptosis by GM-CSF compared with those in apoptotic TF-1 cells. Because ceramide has been shown to affect PKCα subcellular distribution, these data implicate a role for ceramide in mediating the rapid postirradiation translocation and inhibition of PKCα in TF-1 cells not rescued from apoptosis by GM-CSF. Expression of the antiapoptotic protein Bcl-2 doubled in TF-1 cells rescued from apoptosis by GM-CSF, but did not increase in unrescued cells. Our findings suggest that activated PKCα and increased expression of Bcl-2 after γ irradiation determine survival in TF-1 cells rescued from apoptosis with GM-CSF and that PKCδ plays a role in mediating signals involved in sensing cellular damage and/or regulation of cell damage repair.

scholarly journals Truncated PPM1D Prevents Apoptosis in the Murine Thymus and Promotes Ionizing Radiation-Induced Lymphoma

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2068
Author(s):  
Andra S. Martinikova ◽  
Monika Burocziova ◽  
Miroslav Stoyanov ◽  
Libor Macurek
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Tumor Formation ◽  
Cell Cycle Checkpoints ◽  
Negative Regulator ◽  
Tumor Suppressor P53 ◽  
Mouse Thymus ◽  
Truncating Mutation ◽  
T Cell Lymphomas ◽  
Radiation Induced

Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/− mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.

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