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E Dikomey

Publications and source records attributed to E Dikomey.

At least 19 recordsLinked to original sources
PubMed1999

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts.

The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5-7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 GY, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant.

Antigens, Nuclear↗
PubMed1999

Overexpression of human Ku70/Ku80 in rat cells resulting in reduced DSB repair capacity with appropriate increase in cell radiosensitivity but with no effect on cell recovery.

The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80. The overexpression was found to result in a 20% reduction of the DNA-PK activity. The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min). However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics. In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%). A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%). R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique. Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs. The two cell lines, however, did not vary in cell recovery. For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios. These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery.

Animals↗
PubMed1998

Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines.

PURPOSE: To test the relationship between cell killing and non-repaired DNA strand breaks both in repair proficient and deficient cell lines. MATERIALS AND METHODS: Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs1, xrs5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis. RESULTS: The nine cell lines showed a broad spectrum in radiosensitivity with SF2 values ranging from 0.018 to 0.58. The cell lines did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r2=0.29) but a fairly good correlation for double-strand breaks (r2=0.87). This correlation was found to hold both for repair proficient and deficient cell lines. CONCLUSIONS: The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24h after irradiation can be used as an indicator of cellular radiosensitivity.

Animals↗
PubMed1998

DNA-repair, cell killing and normal tissue damage.

BACKGROUND: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. MATERIAL AND METHODS: Review. RESULTS: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. CONCLUSION: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly.

Animals↗
PubMed1997

Relationship between PCC fragments and cell killing studied in X-irradiated CHO, CHO-K1 cells and two radiosensitive mutants xrs1 and xrs5.

PURPOSE: To investigate the correlation between PCC fragments and cell killing. MATERIALS AND METHODS: Induction and repair of DNA fragments were measured in CHO, CHO-K1, xrs1 and xrs5 cells using the premature chromosome condensation (PCC) technique and cell survival was determined by a colony assay. RESULTS: The number of PCC fragments measured in cells immediately fused after X-irradiation was the same for CHO (3.4 +/- 0.16/cell/Gy) and CHO-K1 (3.6 +/- 0.12/cell/Gy) cells but significantly higher for xrs1 (4.9 +/- 0.07/cell/Gy) and xrs5 cells (7.0 +/- 0.4/cell/Gy). The repair curve of PCC fragments studied for CHO, CHO-K1 and xrs5 cells was best described by a monophasic exponential decline with a final plateau; the half-time of this decline was always about 30 min. The number of unrejoined PCC fragments, which was measured 14h after irradiation, increased linearly with dose. The steepest increase was found for xrs5 cells (5.5 +/- 0.3 fragments per cell and per Gy), the lowest for CHO/CHO-K1 (0.9 +/- 0.1; 1.0 +/- 0.1) and for xrs1 in between (3.3 +/- 0.1). For all four cell lines the relationship between cell killing and unrejoined fragments could be described by a single curve with a D0 of 2.5 +/- 0.4 unrejoined PCC fragments per lethal event. CONCLUSIONS: The data showed that the number of unrejoined PCC fragments can be used as an indicator of cellular radiosensitivity.

Animals↗
PubMed1997

Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line.

The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 degrees C, 0.5 h) separated by time intervals up to 8 h. DNA dsb were measured by PFGE and survival by the colony forming assay. In non-heated HeLa S3 cells repair of dsb was biphasic with the majority of breaks being repaired fast with a half-time of 14 min and only a minority were repaired slowly with a half-time of 130 min. Heat applied immediately after irradiation was found to cause an increase in both half-times but mainly to result in an increased fraction of slowly repairable dsb. The latter effect was shown to result from the formation of additional dsb. The number of additional dsb declined when irradiation and heat were separated by an interval at 37 degrees C with a half-time of 120 +/- 30 min. This half-time was similar to the half-time of 100 +/- 20 min found for the loss of thermal radiosensitization studied for the same protocol. Both processes were recently found also to correlate in CHO cells but occurred much faster in rodent cells than in the human HeLa S3 cells used in the current study. These results show that in human cells, unlike previously suggested on the basis of rodent cells, thermal radiosensitization is still a substantial contributor to the killing efficacy of a combined treatment even when irradiation and heat are separated by a time internal of 4 h.

Cell Survival↗
PubMed1997

Heat effects on the repair of DNA double-strand breaks in CHO cells.

The effect of heat (43-45 degrees C) on the induction and the repair of DNA double-strand breaks (dsb) was studied in CHO cells after 60 Gy of X-rays using constant-field gel electrophoresis. Heat given prior to irradiation was found not to alter the number of dsb when measured immediately after irradiation. In non-heated cells, about 80% of all dsb were rapidly repaired with a half-time of 4 min, while 20% were repaired more slowly with T4 = 160 min. These kinetics were grossly altered by heat. Both the fast and the slow process were retarded. However, the main effect of heat was an increase in the number of slowly rejoined dsb. This increase was shown to result from the additional formation (up to 1.4-fold the initial number) of dsb early during the repair course. It is suggested that the additional dsb arose from base damage, the repair of which was unbalanced by heat. No evidence was found for apoptosis being involved in this process. The kinetics of the additional dsb was found to correlate with thermal radiosensitization.

Animals↗
PubMed1996

Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis.

Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60 Gy. The repair curve obtained was biphasic with the respective half-times of tau 1 = 3.8 +/- 0.9 and tau 2 = 118 +/- 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells nonreparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenetic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial deletions). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments < or = 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells.

Animals↗
PubMed1995

Separation of DNA fragments induced by ionizing irradiation using a graded-field gel electrophoresis.

A method is described that allows a separation of X-ray induced DNA fragments by graded-field gel electrophoresis. Synchronized G1 and asynchronous CHO cells were embedded in agarose and irradiated with X-ray doses ranging from 1 to 100 Gy. Following proteolysis by sarcosine and proteinase K, electrophoresis was run for 49 h using graded electric fields with stepwise increasing field strength (0.6, 1.5, 3 and 9 V/cm). Since the molecular size of DNA able to migrate decreased with increasing voltage, each voltage step led to the generation of a distinct DNA band with the largest fragments in band 1, fragments of intermediate size in bands 2 and 3 and the smallest fragments in band 4. Using yeast chromosomal DNA as a reference, the molecular weight of eluted fragments was calculated to range from 1 to 10 Mbp. It could be shown that the fragment size was not the only criterion that discriminates migrating from non-migrating DNA. DNA fragments were found to be retained in the well by an unknown factor presumably associated with DNA conformation. This retention factor increased with increasing fragment size. Graded-field gel electrophoresis also allowed the determination of the absolute number of double-stranded breaks (dsb) induced, which amounted to 11.5 x 10(-12) dsbs Gy-1 Da-1 corresponding to 37 dsbs/G1 cell.

Animals↗
PubMed1995

Comparison between the alkaline unwinding technique and neutral filter elution using CHO, V79 and EAT cells.

Induction and repair of DNA strand breaks were measured in X-irradiated CHO, V79 and EAT cells using either the alkaline unwinding or the neutral filter elution techniques. After irradiation on ice, the cells were incubated at 37 degrees C for various times to allow for repair. The repair curves obtained were quite similar and the fast initial decline was always characterized by a half-time of 6-8 min and the final slow phase by a half-time of about 200 min independently of the technique used. The curves were found to differ only in the relative fraction of damage repaired during the fast or slow phase. From the similarity of the half-times it is concluded that the fast phase recorded by alkaline unwinding reflects the repair of single-strand breaks but also the repair of fast rejoining double-strand breaks. Comparing the known ratio of induced single- and double-strand breaks with the measured ratio of fast and slowly rejoining strand breaks as derived from the kinetics, it can be further concluded that the slow phase measured by alkaline unwinding covers the repair of both slowly rejoining double strand breaks as well as slowly rejoining single strand lesions.

Animals↗
PubMed1995

Comparative studies of induction and repair of DNA double-strand breaks in X-irradiated alveolar macrophages and resting peripheral blood lymphocytes using constant-field gel electrophoresis.

Induction and repair of X-ray-induced DNA double-strand breaks (dsbs) was compared for normal broncho-alveolar macrophages and human peripheral blood lymphocytes, using CHO cells as a reference cell model. The cells, upon their separation, were processed in a similar manner. After X-irradiation, cell lysis and proteinase K treatment, the DNA samples were subjected to constant-field gel electrophoresis (CFGE) followed by fluorimetric densitometry for quantification of released DNA. Induction of dsbs after X-ray doses of 5-100 Gy was found to show no gross differences for all cell systems used. Repair of dsbs was studied after X-ray dose of 60 Gy for up to 24 h after irradiation. The repair curves obtained proved to be similar for bronchoalveolar macrophages and CHO cells (97% of all dsbs rejoined after 24 h). However, in blood lymphocytes from normal subjects and from bone marrow recipients, dsb repair proceeded rapidly only for 0.5-1 h post-irradiation, being followed by the gradual degradation of DNA at longer intervals. The kinetics of DNA degradation correlated with cytological features of pyknosis and necrosis.

Animals↗
PubMed1995

Correlation between thermal radiosensitization and slowly rejoined DNA strand breaks in CHO cells.

The effect on the repair of slowly rejoined strand breaks was studied in CHO cells using the alkaline unwinding technique. Heat (45 degrees C, 20 min) combined with a X-ray dose of 9 Gy was found to result in an increased half-time of repair but also in an increased number of slowly rejoined strand breaks. When a time interval at 37 degrees C was inserted between irradiation and heat, the half-time of repair was not altered, whereas the number of slowly rejoined strand breaks as measured 300 min after irradiation decreased with increasing time interval between the two treatments. The half-time of 18 +/- 2 min suggested that the additionally formed, slowly rejoined strand breaks arise from a certain type of radiation-induced DNA base lesions with repair of which is modified by heat. The effect of X-irradiation combined with heat was also studied for cell survival. When irradiation and heat were separated by an incubation at 37 degrees C, cell survival increased with a half-time of 20 +/- 2 min, which is similar to that measured for the number of additional, slowly rejoined strand breaks. For a great variety of combined treatments, the reduction in cell survival correlates well with the enhanced number of slowly rejoined strand breaks measured 300 min after irradiation. This positive correlation and the similarity in the half-times mentioned above suggests that thermal radiosensitization results from the number of additional, slowly rejoined strand breaks formed when irradiation was combined with heat.

Animals↗
PubMed1994

Relationship between non-reparable DNA strand breaks and cell survival studied in X-irradiated CHO, CHO K1, xrs1 and xrs5 cells.

Non-reparable DNA strand breakage was measured by means of alkaline unwinding technique in CHO, CHO K1, xrs1 and xrs5 cells for X-ray doses ranging from 15 to 180 Gy. For comparison, cell survival was recorded for doses up to 9 Gy using the colony forming assay. DNA strand breaks determined 24 h after irradiation were considered as non-preparable, since it was shown previously that repair processes are completed by 20 h after irradiation and the level reached remained constant for at least another 10 h. The number of non-reparable strand breaks was found to increase with dose. This increase was much steeper for xrs1 and xrs5 cells as compared with that for the parental strain CHO K1. This difference correlates with the respective cellular radiosensitivities. For the three cell lines the ratio of non-reparable strand break/lethal event is about 15:1. For the original CHO cells, which showed the same radiosensitivity as K1 cells, much less non-reparable breaks were found when compared with K1 cells. For CHO cells the ratio of residual strand breaks/lethal event is about 1:1. From these data it is concluded that about one non-reparable DNA strand break is sufficient for cell kill, and that the higher number of non-reparable breaks found for CHO K1 cells and xrs mutants results from unrejoined breaks which do not affect lethality. These breaks might result from DNA degradation.

Animals↗
PubMed1994

Non-reparable DNA strand breaks and cell killing studied in CHO cells after X-irradiation at different passage numbers.

Non-reparable DNA strand breaks were measured in X-irradiated CHO cells by means of the alkaline unwinding technique and were compared with cell survival measured by the colony assay. The experiments were performed with cells at passage numbers 10, 50 and 110 after thawing from stock culture. Cellular radiosensitivity was found to be identical for all three passage numbers used. By contrast, the dose-response of non-reparable DNA strand breaks was only the same for passage numbers 10 and 50 but significantly steeper for cells irradiated in passage number 110. The ratio of non-reparable breaks to lethal events, as calculated from the survival curves, was found close to 1:1 for cells irradiated at passage numbers 10 and 50 but increased to 20:1 at passage number 110. These data indicate that the number of non-reparable strand breaks measured after irradiation not only depends on cellular radiosensitivity but also on other parameters such as the age of the cell culture.

Animals↗
PubMed1994

Induction and repair of DNA base damage studied in X-irradiated CHO cells using the M. luteus extract.

DNA base damage was measured in Chinese hamster ovary cells X-irradiated under aerobic conditions using an extract of the bacterium Micrococcus luteus. The glycosylases and endonucleases present in this extract recognize damaged bases and convert them into strand breaks (termed endonuclease-sensitive sites, enss). Strand breaks were detected by the alkaline unwinding technique. The induction of enss was measured for X-ray doses ranging up to 45 Gy. The relative frequency of all enss related to all radiation induced strand breaks was 1.7 +/- 0.4. Repair of enss was studied for a radiation dose of 45 Gy. The number of enss was found to decrease exponentially with time after irradiation with a half-time of tau enss = 37 +/- 8 min. The repair kinetics that were also measured for all X-ray-induced DNA strand breaks were found to consist of three phases: fast, intermediate and slow. The intermediate phase was fitted under the assumption that this phase results from the formation and repair of secondary single-strand breaks generated by enzymatic incision at the sites of base damage repair. The relative frequency of base damage derived from this fit was 1.8 +/- 0.5 and the half-time of base damage repair was tau in = 32 +/- 6 min. The agreement of this half-time with the half-time obtained when base damage was measured directly using the M. luteus assay gives support to the interpretation that the intermediate phase of the total repair curve represents the kinetics of secondary strand breaks resulting from base damage by enzymatic incision.

Animals↗
PubMed1993

Correlation between thermal radiosensitization and heat-induced loss of DNA polymerase beta activity in CHO cells.

The increase in radiosensitivity caused by various kinds of single and combined heat treatments was studied in CHO cells and related to the heat-induced loss of the DNA polymerase beta activity. Thermal radiosensitization was quantified by the thermal enhancement ratio TER10% determined on the 10% survival level and by the parameters alpha and beta obtained from fitting cellular survival data to the equation -ln(S/S0) = alpha D + beta D2. The values for TER10% and the alpha-term showed only a poor correlation with the inverse of polymerase beta activity; the data for single heating at temperatures exceeding 41.5 degrees C and the data for thermotolerant cells fell on the same straight line, whereas for single heating at T < or = 41.5 degrees C and for cells exposed to a high-to-low temperature sequence (step-down heating) the increase in TER10% and alpha was much steeper than found for the other heat treatments. By contrast, a linear relationship was shown to exist between the beta-term of cellular radiosensitivity and the loss of polymerase beta activity as expressed by the reciprocal value of the enzyme activity. This relationship was the same for all kinds of heat treatments applied, suggesting that for CHO cells the increase in the beta-term observed after combined treatment with heat and radiation might be causally related to the heat-induced loss of polymerase beta activity.

Animals↗