Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle.

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.

Computerized video time-lapse microscopy studies of ionizing radiation-induced rapid-interphase and mitosis-related apoptosis in lymphoid cells.

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.

Using computerized video time lapse for quantifying cell death of X-irradiated rat embryo cells transfected with c-myc or c-Ha-ras.

Rat embryo fibroblasts that had been transfected with the c-myc or c-Ha-ras oncogene were X-irradiated, after which individual cells and their progeny were followed in multiple fields for 5-6 days by computerized video time lapse microscopy to quantify the lethal events that resulted in loss of clonogenic survival. The loss of clonogenic survival of X-irradiated (9.5 or 2.5 Gy) REC:myc cells was attributed almost entirely to the cells dying by apoptosis, with almost all of the apoptosis occurring after the progeny had divided from one to four times. In contrast, the loss of clonogenic survival of X-irradiated REC:ras cells was attributed to two processes. After 9.5 Gy, approximately approximately 60% of the nonclonogenic cells died by apoptosis (with a very small amount of necrosis), and the other 40% underwent a senescent-type process in which some of the cells and their progeny stopped dividing but remained as viable cells throughout 140 h of observation. Both processes usually occurred after the cells had divided and continued to occur in the cells' progeny for up to five divisions after irradiation. Furthermore, the duration of the apoptotic process was shorter for REC:myc cells (0.5-1 h) than for REC:ras cells (4-5 h). By using computerized video time lapse to follow individual cells, we were able to determine the mode of cell death. This cannot be determined by conventional clonogenic survival experiments. Also, only by following the individual cells and their progeny can the true amount of apoptosis be determined. The cumulative percentage of apoptosis scored in whole populations, without distinguishing between the progeny of individually irradiated cells, does not reflect the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results because both clonogenic and nonclonogenic cells are dividing as nonclonogenic cells are apoptosing or senescing over a period of many days. For example, after 9.5 Gy, which causes reproductive cell death in 99% of both types of cells, the cumulative percentage of the cells scored as dead in the whole population at 60- 80 h after irradiation, when the maximum amount of cumulative apoptosis occurred, was approximately 60% for REC:myc cells, compared with only approximately 40% for REC:ras cells.

Selection and sequencing of interchromosomal rearrangements from gamma-irradiated normal human fibroblasts.

PURPOSE: To determine the sequences that flank sites of interchromosomal DNA rearrangements and to determine the relative frequency of inter- and intrachromosomal rearrangements induced by 30 Gy gamma-irradiation in a region 5' from exon I of the c-myc gene in normal human fibroblasts (IMR-90). MATERIALS AND METHODS: A modification of an inverse polymerase chain reaction (PCR) procedure, developed previously to detect rearrangements, was used. Inverse PCR products were re-amplified using primers designed to determine whether the product was a result of an inter- or intrachromosomal rearrangement. Possible interchromosomal rearrangements were then sequenced. RESULTS AND CONCLUSIONS: Four of 12 different products analyzed were potentially derived from interchromosomal rearrangements, while the remainder derived from intrachromosomal rearrangements. For three of the potential interchromosomal rearrangements, the sequence recombining with c-myc was unidentified, while in the other case the sequence was homologous to an L1 element. The frequencies of inter- and intrachromosomal rearrangements induced by 30 Gy gamma-irradiation in a 2 kbp region flanking the c-myc gene of IMR-90 cells were calculated to be at least 1.6x10(-4) and 3.3x10(-4) respectively. No clear association between sequence context and sites of radiation-induced rearrangement was found; however, two of the four sequenced rearrangements involved breakpoints in the 5'-flanking region of c-myc that occurred immediately after the sequence AAAGG.

Detection and sequencing of ionizing radiation-induced DNA rearrangements using the inverse polymerase chain reaction.

PURPOSE: To develop a procedure, using the inverse polymerase chain reaction, to detect and sequence ionizing radiation-induced DNA rearrangements without prior phenotypic selection of mutant cells. METHOD: Normal human fibroblast cells (IMR-90) were given 30Gy of gamma-irradiation and then incubated at 37 degrees C for 23h to allow DNA repair. Rearrangements of the sequence 5' to the c-myc gene were examined by amplifying the region using inverse PCR followed by DNA sequencing. RESULTS: Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for repair than were obtained from cells that were either unirradiated or were irradiated and then lysed immediately. PCR products from seven putative radiation-induced DNA rearrangements were sequenced. Of these products, one contained an unidentified sequence (a possible inter-chromosomal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangements). The sequencing data suggested that the sites of DNA rearrangement breakpoints were non-randomly distributed and possibly associated with topoisomerase I consensus cleavage sequences. There was a significant level of direct homology between the sequences flanking the breakpoints. CONCLUSIONS: The procedure developed was able to detect both inter- and intra-chromosomal rearrangements.

Persistent decrease in viability as a function of X irradiation of human bladder carcinoma cells in G1 or S phase.

A persistent decrease in viability after treatment with a variety of mutagenic agents has been observed previously, but the dependence of the decrease on the phase of the cell cycle in which the cells are treated has not been fully explored. Synchronous human bladder carcinoma cells (EJ30-15) were obtained by mitotic selection (88-96% in or near mitosis). As monitored by microscopy and pulse labeling with [3H]dThd, approximately 98% of the cells were in G1 phase when they were irradiated after 3 h of incubation, and approximately 80% were in S phase when they were irradiated after 14 h of incubation. The initial plating efficiencies demonstrated no difference in cell survival when cells were irradiated in G1 or S phase, with normalized clonogenic survival and standard error of 60+/-6% for 3 Gy and 13+/-2% for 6 Gy. However, when the cell populations were allowed to incubate and were replated 5 to 33 days later (5.5 to 36 doublings), a difference between the populations irradiated in G1 and S phase became clear. Cells that were irradiated with 6 Gy regained and maintained the high plating efficiencies (67.9+/-3.6%) of the unirradiated populations much sooner when they were irradiated in S phase compared with irradiation in G1 phase, i.e. 11 days (12 cell doublings) for S phase compared to approximately 20 days (22 cell doublings) for G1 phase. During these periods when the plating efficiencies were increasing, the populations irradiated in G1 phase were multiplying at rates lower than those for the populations irradiated in S phase. Furthermore, after 6 Gy, more giant cells and multinucleated cells were seen in the populations irradiated in G1 phase than in the populations irradiated in S phase. These results indicate that, although the clonogenic survival was the same for cells irradiated in G1 or S phase, the residual damage in progeny of the irradiated cells persisted longer (approximately 20 days compared to 11 days) when cells were irradiated in G1 phase than when they were irradiated in S phase.

Comparisons of the frequencies and molecular spectra of HPRT mutants when human cancer cells were X-irradiated during G1 or S phase.

In an attempt to elucidate mechanisms underlying the variation in radiosensitivity during the cell cycle, mutations in the HPRT gene were selected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G1 or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with approximately 98% of the cells in G1 phase when they were irradiated after 3 h of incubation, and 75% in S phase when they were irradiated after 14 h of incubation. The mutant frequencies were approximately 4-fold higher (P < 0.01) when cells were irradiated in G1 phase compared with S phase, and the lowest frequency (1.5 x 10(-5) for 3 Gy during S phase) was approximately 10-fold higher than the spontaneous frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base-pair changes or small deletions less than 20 bp; class 2, which consisted of deletions greater than 20 bp but with one or more HPRT exons present; and class 3, which consisted of deletions encompassing the entire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A "hotspot" for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G1 phase, the percentages for the different classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontaneous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells were irradiated in G1 phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase from 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hypothesize that these differences occur because: (a) there is more error-free repair of double-strand breaks (DSBs) during S than G1 phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in each approximately 1-Mbp region 5' and 3' of the gene, cause a class 3 mutation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations.