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2023 Alteration of cellular radiation response as a consequence of defective DNA mismatch repair
252
I. .I. Radiation
Oncology*Biology*Physics
Volume
39, Number
2, Supplement,
1997
2023 ALTERATION Theodore Department Baltimore,
OF CELLULAR L. DeWeese”,
RADIATION
Jennifer
RESPONSE
Nieok
M. Bueci’,
AS A CONSEQUENCE
A. Larrier’,
Richard
of ‘Oncology, Division of Radiation Oncology and Department Maryland; ‘Genox Corporation, Baltimore, Maryland; “The
G. Cutler’, of ‘Urology, Netherlands
OF DEFECTIVE Hein
te Riele”,
DNA William
MISMATCH
REPAIR
G. Nelson’*
The Johns Hopkins University, Cancer Institute, Amsterdam,
School of Medicine, The Netherlands.
A number of genes have been implicated in the response of mammalian cells to ionizing radiation. Among these include the genes M3 and PZI. Disruption of these genes can alter the predicted cellular behavior following radiation-induced DNA damage. Similarly, cells defective in mismatch repair are known to be tolerant to the lethal effects of alkylating agents. We hypothesized that mammalian cells which are defective in mismatch repair and tolerant to alkylating DNA damage might also be tolerant to the effects of oxidative DNA damage inflicted by ionizing radiation. Purpose/Objective:
Moose embryonic stem cells homozygous for disrupted Msh2 alleles (Msh2-A), heterozygous for a disrupted Msh2 allele & Methods: or intact cells (MshZ+l+) were exposed to both acute dose (1 Gy/min) and low dose rate (LDR) radiation (0.004 Gy/min) and cell survival was determined by clonogenic assay. Apoptosis induced by LDR was assessed by a terminal transferase assay. Immunoblot analysis was performed in order to evaluate induction of the polypeptides p53 and ~21. Another measnre of radiation damage tolerance may be accumulation of oxidative DNA species. Therefore, we monitored levels of 8-hydroxyguanine (8-OHG) and 8-hydroxyadenine (8-OHA) by gas chromatography - mass spectrometry with selected ion monitoring (GC-MS/SIM). Materials (MshZ+l-)
Cells containing either one or two disrupted Msh2 alleles (MshZ+i-, M&Z-l-) were found to be less sensitive to LDR than cells containing a complete complement of Msh2 alleles (Msh2+/+). Interestingly, all three cell lines had a nearly identical radiosensitivity to acute dose ionizing radiation despite differences in mismatch repair capacity. Apoptosis after LDR also varied between cells, with the Msh2+/+ cells exhibiting higher levels of apoptosis as compared to either the Msh2+/- or Msh2-l- cell lines. In addition, GC-MS/SIM revealed the Msh2+/- and MshZ-l- cell lines to have an approximately ten fold greater accnmulation of the radiation-induced oxidative DNA species 8-OHG and 8-OHA as compared to the MshZ+/+ cell line.
Results:
These data reveal that mammalian cells containing disrupted A4sh2 alleles display an increased radioresistance as well as decreased Conclusion: radiation-induced apoptosis after LDR exposure, despite a similar pattern of p53 induction. These same mismatch repair defective cells are also able to accumulate oxidative DNA damage. Taken together, these data suggest that mismatch repair may be crucial in the recognition and/or repair of radiation-induced oxidative DNA damage.
2024 IN V7TRO IRRADIATION ACTIYITY
Scott P. Lankford’,
OF EARLY-PASSAGE
Fady IL Geara’,
Department cd Radiation Hwstoa, Texas 77030.
Oncology’
and William
FIUMAN
FIIIROBLASTS
INDUCES
SENESCENCE-ASSOCIATED
@-GALACTOSIDASE
A. Brwk’
aad Department
of Experimental
Radiation
Oocalo&,
The University
of Texas, M.D. Anderson
Csacer
Center,
Purpose
ionizing radiation, like other cytotox~c agents, has been shown lo induce morphologic changes characteristic of cellular senescencein human dtploid tibroblasts. Whether-or-not these changes are associated with senescence-spxitic gene expression was examined by meawing the expression of senescenceassociatedS-galactosidase(SA S-pal) in irradiated early-passage human flareblasts. SA p-pal has an optimum pH of 6.0 and has been shown recently 10be a specific bloararker of seaescentcells in both cultured human fibmblasts and in human skin. Methods aad Materials Early-passage human tibroblasts were plated (40-50 cells per plate) and then treated with incremental doses of ionizing radiation (0, 2,4, or 6 Gy). Cells were incubated for 1 day, 7 days, or 14 days following treatment, and were then fixed and stained for SA p-gal. Each plate was scored using the 16-tell colony assay, in which small, arrested colonies of 1-15 cells and growing colonies of > 16 cells were mnnted as distinct events. The phenotyp of each colony was swrcd using a stereo micmscclx as SA p-gal positive (i.e., senescentphenotype), SA p-gal negative. or equiwxal. The percentage of SA p-gal positive colooies was calculated and comparisons were made for each dose of irradiation and for each inculxtion time. There was a statistically significant increase in the percentage of SA B-gal positive colooies in the rrradiated group compared to okradiated cDntmls This increase was visible from day 1 and perusled 14 days following treatment. On day 1, the SA p-gal poaltive percentage was 7.8 f 4.2 (mean + SEM) in the contml group compared to 30.9 + 3.5 in the irradiated group (p = 0.01, Kendall correlation). There was no detectable dose-effect on the induction of SA pgal positivity (2 Gy = 30.0 + 7.5; 4 Gy = 38.6 + 4.3; 6 Gy = 24.2 + 4.0; p = 0.3). On day 14, the SA p-gal positive percentage was 30.4 + 2.6 in the ccntml gmop compared to 65.9 2 2.8 in the imadialed group (p < 0.001). Differences between individual doses of irradiation were borderline significant (2 Gy = 59.6 + 5.7; 4 Gy = 64.9 2 2.6,6 Gy = 73.2 + 4.6; p = 0.06). Similarly, there was a signiticantly hiSher prcenta& of -z 16 cell colonies (i.e., Srowth arrested) in
the irradiated group than in the control group on day 14 (93.8 + 1.4 vs. 60.0 + 2.5; p < 0.001) and there was a significant increase in the percentage of < 16 cell colonies with mcreasing dosesof radiation (p < 0.001). Conclusioar There was a statistically significant and prolonged increasein SA p-gal expression in irradiated normal fib&lasts compared to onirradiated controls, supporting the hypothesis that ionizing radiation induces or acceleratescellular senescencein viho. The possibility that radiothera~ induces SA S-pal
expression
in human skin is currently
under investigation.
I. .I. Radiation
Oncology*Biology*Physics
Volume
39, Number
2, Supplement,
1997
2023 ALTERATION Theodore Department Baltimore,
OF CELLULAR L. DeWeese”,
RADIATION
Jennifer
RESPONSE
Nieok
M. Bueci’,
AS A CONSEQUENCE
A. Larrier’,
Richard
of ‘Oncology, Division of Radiation Oncology and Department Maryland; ‘Genox Corporation, Baltimore, Maryland; “The
G. Cutler’, of ‘Urology, Netherlands
OF DEFECTIVE Hein
te Riele”,
DNA William
MISMATCH
REPAIR
G. Nelson’*
The Johns Hopkins University, Cancer Institute, Amsterdam,
School of Medicine, The Netherlands.
A number of genes have been implicated in the response of mammalian cells to ionizing radiation. Among these include the genes M3 and PZI. Disruption of these genes can alter the predicted cellular behavior following radiation-induced DNA damage. Similarly, cells defective in mismatch repair are known to be tolerant to the lethal effects of alkylating agents. We hypothesized that mammalian cells which are defective in mismatch repair and tolerant to alkylating DNA damage might also be tolerant to the effects of oxidative DNA damage inflicted by ionizing radiation. Purpose/Objective:
Moose embryonic stem cells homozygous for disrupted Msh2 alleles (Msh2-A), heterozygous for a disrupted Msh2 allele & Methods: or intact cells (MshZ+l+) were exposed to both acute dose (1 Gy/min) and low dose rate (LDR) radiation (0.004 Gy/min) and cell survival was determined by clonogenic assay. Apoptosis induced by LDR was assessed by a terminal transferase assay. Immunoblot analysis was performed in order to evaluate induction of the polypeptides p53 and ~21. Another measnre of radiation damage tolerance may be accumulation of oxidative DNA species. Therefore, we monitored levels of 8-hydroxyguanine (8-OHG) and 8-hydroxyadenine (8-OHA) by gas chromatography - mass spectrometry with selected ion monitoring (GC-MS/SIM). Materials (MshZ+l-)
Cells containing either one or two disrupted Msh2 alleles (MshZ+i-, M&Z-l-) were found to be less sensitive to LDR than cells containing a complete complement of Msh2 alleles (Msh2+/+). Interestingly, all three cell lines had a nearly identical radiosensitivity to acute dose ionizing radiation despite differences in mismatch repair capacity. Apoptosis after LDR also varied between cells, with the Msh2+/+ cells exhibiting higher levels of apoptosis as compared to either the Msh2+/- or Msh2-l- cell lines. In addition, GC-MS/SIM revealed the Msh2+/- and MshZ-l- cell lines to have an approximately ten fold greater accnmulation of the radiation-induced oxidative DNA species 8-OHG and 8-OHA as compared to the MshZ+/+ cell line.
Results:
These data reveal that mammalian cells containing disrupted A4sh2 alleles display an increased radioresistance as well as decreased Conclusion: radiation-induced apoptosis after LDR exposure, despite a similar pattern of p53 induction. These same mismatch repair defective cells are also able to accumulate oxidative DNA damage. Taken together, these data suggest that mismatch repair may be crucial in the recognition and/or repair of radiation-induced oxidative DNA damage.
2024 IN V7TRO IRRADIATION ACTIYITY
Scott P. Lankford’,
OF EARLY-PASSAGE
Fady IL Geara’,
Department cd Radiation Hwstoa, Texas 77030.
Oncology’
and William
FIUMAN
FIIIROBLASTS
INDUCES
SENESCENCE-ASSOCIATED
@-GALACTOSIDASE
A. Brwk’
aad Department
of Experimental
Radiation
Oocalo&,
The University
of Texas, M.D. Anderson
Csacer
Center,
Purpose
ionizing radiation, like other cytotox~c agents, has been shown lo induce morphologic changes characteristic of cellular senescencein human dtploid tibroblasts. Whether-or-not these changes are associated with senescence-spxitic gene expression was examined by meawing the expression of senescenceassociatedS-galactosidase(SA S-pal) in irradiated early-passage human flareblasts. SA p-pal has an optimum pH of 6.0 and has been shown recently 10be a specific bloararker of seaescentcells in both cultured human fibmblasts and in human skin. Methods aad Materials Early-passage human tibroblasts were plated (40-50 cells per plate) and then treated with incremental doses of ionizing radiation (0, 2,4, or 6 Gy). Cells were incubated for 1 day, 7 days, or 14 days following treatment, and were then fixed and stained for SA p-gal. Each plate was scored using the 16-tell colony assay, in which small, arrested colonies of 1-15 cells and growing colonies of > 16 cells were mnnted as distinct events. The phenotyp of each colony was swrcd using a stereo micmscclx as SA p-gal positive (i.e., senescentphenotype), SA p-gal negative. or equiwxal. The percentage of SA p-gal positive colooies was calculated and comparisons were made for each dose of irradiation and for each inculxtion time. There was a statistically significant increase in the percentage of SA B-gal positive colooies in the rrradiated group compared to okradiated cDntmls This increase was visible from day 1 and perusled 14 days following treatment. On day 1, the SA p-gal poaltive percentage was 7.8 f 4.2 (mean + SEM) in the contml group compared to 30.9 + 3.5 in the irradiated group (p = 0.01, Kendall correlation). There was no detectable dose-effect on the induction of SA pgal positivity (2 Gy = 30.0 + 7.5; 4 Gy = 38.6 + 4.3; 6 Gy = 24.2 + 4.0; p = 0.3). On day 14, the SA p-gal positive percentage was 30.4 + 2.6 in the ccntml gmop compared to 65.9 2 2.8 in the imadialed group (p < 0.001). Differences between individual doses of irradiation were borderline significant (2 Gy = 59.6 + 5.7; 4 Gy = 64.9 2 2.6,6 Gy = 73.2 + 4.6; p = 0.06). Similarly, there was a signiticantly hiSher prcenta& of -z 16 cell colonies (i.e., Srowth arrested) in
the irradiated group than in the control group on day 14 (93.8 + 1.4 vs. 60.0 + 2.5; p < 0.001) and there was a significant increase in the percentage of < 16 cell colonies with mcreasing dosesof radiation (p < 0.001). Conclusioar There was a statistically significant and prolonged increasein SA p-gal expression in irradiated normal fib&lasts compared to onirradiated controls, supporting the hypothesis that ionizing radiation induces or acceleratescellular senescencein viho. The possibility that radiothera~ induces SA S-pal
expression
in human skin is currently
under investigation.