Factors influencing DNA migration from individual cells subjected to gel electrophoresis

Olive, Peggy L.; Wlodek, Danuta; Durand, Ralph E.; Banáth, Judit P.

doi:10.1016/0014-4827(92)90378-l

Cited by 345 publications

(155 citation statements)

References 20 publications

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“…This di erence was similar to that reported in radioresistant and radiosensitive medulloblastoma clones (Tsuboi et al, 1997). In order to investigate the mode of cell death after irradiation we performed assays for apoptosis, using ultrastructural studies, single cell gel electrophoresis (Olive et al, 1991;Kizilian et al, 1999), and annexin V staining. Basal (pre-irradiation) apoptosis was not detectable in S-or AS-IGF1R clones, but after irradiation there was an increase in apoptosis in irradiated AS-IGF1R cells compared with controls (data not shown).…”

Section: Igf1r Downregulation Enhances the Radiosensitivity Of Melanosupporting

confidence: 78%

Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase

et al. 2001

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Section: Igf1r Downregulation Enhances the Radiosensitivity Of Melanosupporting

confidence: 78%

Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase

et al. 2001

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“…DNA dsb repair measured using the neutral comet assay and cellular radiosensitivity were found not to correlate in a study by Olive et al (1992), however one possible explanation for the difference between the present study and that of Olive et al (1992) may reflect the time interval allowed for repair: 4 h in the previous study and 16 h in the present study. It has been noted that measuring residual DNA dsbs using the neutral comet assay at 4 h may underestimate the level of DNA damage as a result of the increase in the percentage of S-phase cells after irradiation, as S-phase DNA migrates inefficiently.…”

Section: Discussioncontrasting

confidence: 87%

“…A linear relationship between dose and initial and residual DNA dsbs was also seen in the majority of the individual experiments for the other five cell lines, albeit with greater variability (correlation coefficients >0.74) (data not shown). In all experiments, the influence of cell age on assay variability was minimized by using confluent cultures to reduce the proportion of cells in S-phase, as the presence of replication forks in DNA can restrict migration under electrophoresis (Olive et al, 1992;. In each experiment, flow cytometry analysis indicated that the proportion of cells in the S-phase of the cell cycle was always below 18% (range 7-17%, mean 12%), irrespective of the cell line used.…”

Section: Resultsmentioning

confidence: 99%

“…A significant correlation has been reported previously between clonogenic radiosensitivity and the levels of initial radiation-induced damage in human tumour cells (Kelland et al, 1988;Nuniiez et al, 1996). It was postulated that this might reflect differences in the number of lesions incurred by the radiosensitive cells compared with the resistant cells, as shown by Peacock et al (1989), or possibly differences in chromatin conformation that affect the accessibility of scavenging molecules to damaged DNA or radical attack (Oleinick et al, 1984;Olive et al, 1992) or the 'presentation' of DNA damage sites (Woudstra et al, 1996). However, the results from this study indicate that in the cervical tumour lines studied here the level of initial DNA damage does not correspond with clonogenic radiosensitivity after HDR and LDR irradiation (Table 3).…”

Section: Discussionmentioning

confidence: 99%

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The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines

Marples

Longhurst²,

Eastham³

et al. 1998

Br J Cancer

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Summary The single-cell gel electrophoresis (comet) assay was used to measure radiation-produced DNA double-strand breaks (dsbs) in a series of seven cervical tumour cell lines (ME180, HT3, C33A, C41, SiHa, MS751 and CaSki). The proportion of DNA dsbs was measured immediately after radiation treatment (initial damage) and 16 h later after incubation at 370C (residual damage). Linear dose-response curves were seen for initial (slopes 0.23-0.66) and residual (slopes 0.16-0.87) DNA dsbs. Neither of the slopes of the linear regression analysis on the initial and on the residual DNA dsbs dose-response curves (range 0-80 Gy) correlated with SF2 (surviving fraction at 2 Gy) measured after high-(HDR) or low-dose-rate (LDR) irradiation. An association was evident between SF2 after HDR and LDR irradiation and the ratio of the absolute level of initial and residual damage after a single dose of 60 Gy. However, a significant correlation was found between HDR (r= -0.78, P = 0.04) and LDR (r = -0.86, P = 0.03) SF2 values and the ratio of the slopes of the initial and residual DNA dsbs dose-response curves (range 0.47-0.99), representing the fraction of DNA damage remaining. These results indicate that the neutral comet assay can be used to predict radiosensitivity of cervical tumour cell lines by assessing the ratio of initial and residual DNA dsbs.

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“…The procedure (Olive et al, 1991) was modified as follows. Lymphocytes were seeded in a 24-well plate (1 Â 10 5 for stimulated and 3 Â 10 5 for nonstimulated) and treated 24 h later with 10 mM MNNG.…”

Section: Determination Of Dsbsmentioning

confidence: 99%

Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1

2004

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Various tumor-therapeutic drugs and environmental carcinogens alkylate DNA inducing O 6 -methylguanine (O 6 MeG) that provokes cell death by apoptosis. In rodent fibroblasts, apoptosis triggered by O 6 MeG is executed via the mitochondrial damage pathway. Conversion of O 6 MeG into critical downstream lesions requires mismatch repair (MMR). This is thought to signal apoptosis upon binding to O 6 MeG lesions mispaired with thymine. Alternatively, O 6 MeG lesions might be processed by MMR giving rise to DNA double-strand breaks (DSBs) during replication that finally provoke apoptosis. To test this, we examined apoptosis triggered by O 6 MeG in human peripheral lymphocytes in which O 6 -methylguanine-DNA methyltransferase (MGMT) had been inactivated by O 6 -benzylguanine (O 6 BG) and which were not proliferating or proliferating upon CD3/CD28 stimulation. Treatment with N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) or the anticancer drug temozolomide induced apoptosis only in proliferating, but not resting cells. With exceptional high alkylation doses (X15 lM of MNNG), apoptosis was also observed in resting lymphocytes, albeit at a lower level than in proliferating cells. This response was not affected by O 6 BG, suggesting that replication-independent apoptosis at high dose levels is caused by lesions other than O 6 MeG. O 6 MeG-triggered apoptosis in proliferating lymphocytes was preceded by a wave of DSBs, which coincided with p53 and Fas receptor upregulation, while Fas ligand, Bax and Bcl-2 expression was not altered. Treatment with anti-Fas neutralizing antibody attenuated MNNG-induced apoptosis in MGMT-depleted proliferating lymphocytes. The data suggest that O 6 MeG is converted by MMR and DNA replication into DSBs that trigger apoptosis by p53 stabilization and Fas/CD95/Apo-1 upregulation. This is supported by the finding that ionizing radiation, inducing DSBs on its own, provokes apoptosis in lymphocytes in a replication-independent way. The strict proliferation dependence of apoptosis triggered by O 6 MeG may explain the specific killing response of MGMT-deficient proliferating cells, including tumors, to O 6 MeG generating anticancer drugs and suggests that tumor proliferation rate, Fas responsiveness, MGMT and MMR status are important prognosis parameters.

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Factors influencing DNA migration from individual cells subjected to gel electrophoresis

Cited by 345 publications

References 20 publications

Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase

Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase

The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines

Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1

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