skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION

Abstract

In this study, we investigated γH2AXser139 and 53BP1ser25, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infi nite lifespan, and a subtype of HMEC 184 (184V) with a fi nite lifespan. Cells were irradiated with 50cGy X-rays, fi xed with 4% paraformaldehyde after 1 hour repair at 37°C, and processed through immunofl uorescence. Cells were visualized with a fl uorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cells exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. The dose and time course will be expanded in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is “normal” and to evaluate the usefulness of cell lines as experimental models.

Authors:
; ; ; ;
Publication Date:
Research Org.:
DOESC (USDOE Office of Science (SC) (United States))
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1052057
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Undergraduate Research; Journal Volume: 7
Country of Publication:
United States
Language:
English

Citation Formats

Wisnewski, C.L., Bjornstad, K.A., Rosen, C.J., Chang, P.Y., and Blakely, E.A. A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION. United States: N. p., 2007. Web.
Wisnewski, C.L., Bjornstad, K.A., Rosen, C.J., Chang, P.Y., & Blakely, E.A. A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION. United States.
Wisnewski, C.L., Bjornstad, K.A., Rosen, C.J., Chang, P.Y., and Blakely, E.A. Mon . "A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION". United States. doi:. https://www.osti.gov/servlets/purl/1052057.
@article{osti_1052057,
title = {A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION},
author = {Wisnewski, C.L. and Bjornstad, K.A. and Rosen, C.J. and Chang, P.Y. and Blakely, E.A.},
abstractNote = {In this study, we investigated γH2AXser139 and 53BP1ser25, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infi nite lifespan, and a subtype of HMEC 184 (184V) with a fi nite lifespan. Cells were irradiated with 50cGy X-rays, fi xed with 4% paraformaldehyde after 1 hour repair at 37°C, and processed through immunofl uorescence. Cells were visualized with a fl uorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cells exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. The dose and time course will be expanded in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is “normal” and to evaluate the usefulness of cell lines as experimental models.},
doi = {},
journal = {Journal of Undergraduate Research},
number = ,
volume = 7,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • In this study, we investigated {gamma}H2AX{sup ser139} and 53BP1{sup ser25}, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infinite lifespan, and a subtype of HMEC 184 (184V) with a finite lifespan. Cells were irradiated with 50 cGy X-rays, fixed with 4% paraformaldehyde after 1 hour repair at 37 C, and processed through immunofluorescence. Cells were visualized with a fluorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cellsmore » exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. We will expand the dose and time course in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is 'normal' and to evaluate the usefulness of cell lines as experimental models.« less
  • In vitro exposure of a human testicular teratoma continuous cell line to fractionated X-irradiation resulted in the expression of resistance to cisplatin. In two independently-derived sublines, designated SUSA-DXR13 and SUSA-DXR10 resulting from treatment with either 13 fractions of 1.5 Gy (dose required to reduce survival by 1 log) or 10 fractions of 3 Gy (dose required to reduce survival by 2 logs) respectively, the IC50 values for cisplatin were 2- and 3.1-fold higher than that of the parental cell line. These sublines were cross-resistant to carboplatin (approximately 2-fold) but not to adriamycin and they showed unaltered radiosensitivities. The SUSA-DXR10 sublinemore » expressed some cross-resistance to mitomycin C and melphalan but none to Carmustine (BCNU). Total glutathione content was significantly reduced in both SUSA-DXR10 and SUSA-DXR13 cells, but the activities of associated enzymes, including the glutathione S-transferases, peroxidase and reductase were not modified significantly in the resistant sublines. Resistance in the SUSA-DXR10 subline was associated with significantly decreased 195mcisplatin uptake (p less than 0.01), but this was not reflected in a reduced level of drug bound to the DNA. The formation and removal of four platinum-DNA adducts were immunochemically quantitated. Immediately following drug treatment there was a higher level of total platination of the DNA in the resistant subline indicative of increased tolerance to DNA damage. After an 18 hr post treatment incubation, there was an indication of some repair capacity in this SUSA-DXR10 cell line, which was not apparent in the parental cells. Neither the parental nor the SUSA-DXR10 cell line was proficient in the repair of the major adduct Pt-GG, whereas both lines repaired the monofunctional adduct and the adduct Pt(GMP)2.« less
  • Yeast and several other organisms are more sensitive to the lethal effects of ionizing irradiation if exposed in the presence of N/sub 2/O as compared to N/sub 2/. It has been suggested that this increased sensitivity is due to the cooperative effects of OH and H/sub 2/O/sub 2/ generated external to the cell wall. Using diploid yeast, wild type for radiation resistance, we have compared the rates of cell death due to ..gamma.. irradiation in N/sub 2/ and N/sub 2/O with the rates of DNA damage measured by gene conversion of trp/sup -/ to trp/sup +/ (a recombinational repair event).more » We find that DNA damage as measured by gene conversion increases at a faster rate, per unit dose, during irradiation in N/sub 2/O as compared to N/sub 2/, just as lethality was higher in N/sub 2/O. When DNA damage was compared in N/sub 2/ and N/sub 2/O at equal levels of survival, however, there was no significant difference between the to irradiation conditions. Therefore, increased lethality during irradiation in N/sub 2/O seems to be directly due to increased DNA damage. If the observed increased lethality results from external OH and H/sub 2/O/sub 2/, the effect of these highly reactive species is expressed by increased internal damage at the level of DNA.« less
  • Two species of turtles that occupy different ecological niches were compared for their usefulness as monitors of freshwater ecosystems where both low-level radioactive and nonradioactive contaminants are present. The pond slider (Trachemys scripta) and common snapping turtle (Chelydra serpentina) were analyzed for the presence of [sup 90]Sr, [sup 137]Cs, [sup 60]Co, and Hg, radionuclides and chemicals known to be present at the contaminated site, and single-strand breaks in liver DNA. The integrity of the DNA was examined by the alkaline unwinding assay, a technique that detects strand breaks as a biological marker of possible exposure to genotoxic agents. This measuremore » of DNA damage was significantly increased in both species of turtles at the contaminated site compared with turtles of the same species at a reference site, and shows that contaminant-exposed populations were under more severe genotoxic stress than those at the reference site. The level of strand breaks observed at the contaminated site was high and in the range reported for other aquatic species exposed to deleterious concentrations of genotoxic agents such as chemicals and ionizing radiation. Statistically significantly higher concentrations of radionuclides and Hg were detected in the turtles from the contaminated area. Mercury concentrations were significantly higher in the more carnivorous snapping turtle compared with the slider; however, both species were effective monitors of the contaminants.« less