Arrhenius relationships from the molecule and cell to the clinic.

There are great differences in heat sensitivity between different cell types and tissues. However, for an isoeffect induced in a specific cell type or tissue by heating for different durations at different temperatures varying from 43-44 degrees C up to about 57 degrees C, the duration of heating must be increased by a factor of about 2 (R value) when the temperature is decreased by 1 degrees C. This same time-temperature relationship has been observed for heat inactivation of proteins, and changing only one amino acid out of 253 can shift the temperature for a given amount of protein denaturation from 46 degrees C to either 43 or 49 degrees C. For cytotoxic temperatures <43-44 degrees C, R for mammalian cells and tissues is about 4-6. Many factors change the absolute heat sensitivity of mammalian cells by about 1 degrees C, but these factors have little effect on Rs, although the transition in R at 43-44 degrees C may be eliminated or shifted by about 1 degrees C. R for heat radiosensitization are similar to those above for heat cytotoxicity, but Rs for heat chemosensitization are much smaller (usually about 1.1-1.2). In practically all of the clinical trials that have been conducted, heat and radiation have been separated by 30-60 min, for which the primary effect should be heat cytotoxicity and not heat radiosensitization. Data are presented showing the clinical application of the thermal isoeffect dose (TID) concept in which different heating protocols for different times at different temperatures are converted into equivalent minutes (equiv) min at 43 degrees C (EM(43)). For several heat treatments in the clinic, the TIDs for each treatment can be added to give a cumulative equiv min at 43 degrees C, namely, CEM(43). This TID concept was applied by Oleson et al. in a retrospective analysis of clinical data, with the intent of using this approach prospectively to guide future clinical studies. Considerations of laboratory data and the large variations in temperature distributions observed in human tumors indicate that thermal tolerance, which has been observed for mammalian cells for both heat killing and heat radiosensitization, probably is not very important in the clinic. However, if thermal tolerance did occur in the clinical trials in which fractionation schemes were varied, it probably would not have been detected because with only the two-three-fold change in treatment time that occurs when comparing one versus two fractions per week, or three versus six total fractions, little difference would be expected in the response of the tumors since both thermal doses were extremely low on the dose-response curve. Data are shown which indicate that in order to test for thermal tolerance in the clinic and to have a successful phase III trial, the thermal dose should be increased about five-fold compared with what has been achieved in previous clinical trials. This increase in thermal dose could be achieved by increasing the temperature about 1.5 degrees C (from 39.5 to 41.0 degrees C in 90% of the tumor) or by increasing the total treatment time about five-fold. The estimate is that 90% of the tumor should receive a cumulative thermal dose (CEM(43)) of at least 25; this is abbreviated as a CEM(43) T(90) of 25. This value of 25 compares with 5 observed by Oleson et al. in their soft tissue sarcoma study. Arguments also are presented that thermal doses much higher than the CEM(43) T(90) induce the hyperthermic damage that causes the tumors to respond, and that the minimum CEM(43) T(90) of 25 only predicts which tumors that receive a certain minimal thermal dose in <90% of the regions of the tumors will respond. For example, in addition to a minimal CEM(43) T(90) of 25 a minimum CEM(43) T(50) of about 400 also may be required for a response. Finally, continuous heating for approximately 2 days at about 41 degrees C during either interstitial low dose-rate irradiation or fractionated high dose-rate irradiation, which we estimate could give a CEM(43) of 75, should be considered in order to enhance heat radiosensitization of the tumor as well as heat cytotoxicity. In order to exploit the use of hyperthermia in the clinic, we need a better understanding of the biology and physiology of heat effects in tumors and various normal tissues. As an example of an approach for mechanistic studies, one specific study is described which demonstrates that damage to the centrosome of CHO cells heated during G(1) causes irregular divisions that result in multinucleated cells that do not continue dividing to form colonies. This may or may not be relevant for heat damage in vivo. However, since normal tissues vary in thermal sensitivity by a factor of 10, similar approaches are needed to describe the fundamental lethal events that occur in the cells comprising the different tissues.

Carcinogenic effects of hyperthermia.

The purpose of this paper is to assess the evidence for and against the premise that hyperthermia is carcinogenic. The paper is one of several published in this issue of the International Journal of Hyperthermia on the subject of the health risks of hyperthermia. The motivation for this issue of the journal was the result of a World Health Organization workshop that dealt with this issue, as it relates to exposure of the population to RF fields. Since hyperthermia can be a natural consequence of such exposures, the health risks of hyperthermia are relevant in this context. Particularly in the case of carcinogenesis, it is necessary to provide a brief overview of the data that have been generated to examine the carcinogenic risks of RF exposure, so that these results can be compared with studies that have examined the carcinogenic risks of hyperthermia. For this reason, the paper is organized into three sections dealing with: (1) effects of heat on DNA damage/repair and mutations, (2) in vivo studies evaluating the carcinogenic potential of heat alone and combined with other carcinogens, and (3) in vivo studies involving RF exposures. The bulk of the data presented indicate that hyperthermia alone is not carcinogenic. If hyperthermia occurs in the presence of exposure to known carcinogens, such as radiation or chemical carcinogens there is the potential for modulation of carcinogenic effects of those agents. In some circumstances, hyperthermia can actually protect against tumour formation. In other instances, hyperthermia clearly increases incidence of tumour formation, but this occurs following thermal exposures (several degrees C temperature rise for up to 1 h or more) and radiation (therapeutic levels as for treatment of cancer) or chemical carcinogen doses higher than would be encountered by the general population. The extrapolation of these results to the general population, where radiation exposure levels would be at background and temperature rise from incidental RF exposure, such as cell phones (which are estimated to cause no more than 0.1 degrees C temperature rise) is not recommended. Current evidence indicates that the temperature elevations resulting from RF exposure are not carcinogenic. Caution should be used in situations where exposure to known carcinogens is combined with thermal exposures high enough to cause tissue damage. A summary of thermal thresholds for tissue damage from hyperthermia is presented in another paper in this special issue (Dewhirst et al.). No data exist that examine the carcinogenic risks of chronic thermal exposures below the threshold for detectable tissue damage, either alone or in combination with known carcinogens. This is an important goal for future research.

Hyperthermic teratogenicity, thermal dose and diagnostic ultrasound during pregnancy: implications of new standards on tissue heating.

Hyperthermia is a recognized teratogen in mammalian laboratory animals and is a suspected teratogen for humans. The purpose of this synopsis is to reanalyse existing data on hyperthermia-induced teratogenic effects in experimental mammalian systems in terms of a thermal dose (temperature:time) concept, and then to illustrate the utility of this concept to human situations involving potential thermal increments to post-implantation embryos and foetuses. For example, the threshold temperature elevation for hyperthermia-induced teratogenic effects in experimental mammals is estimated (but not rigorously tested) to be approximately 1.5 degrees C above core values for exposures of long duration, possibly with a thermal dose of approximately 5 min duration or more at 4 degrees C. This level of tissue temperature increment is within the capability of some modern diagnostic ultrasound (DUS) devices sold within the USA and abroad. Epidemiological studies have not indicated any hazard from the use of DUS, but such studies are limited in sensitivity and were conducted with DUS devices whose acoustic outputs were relatively low compared to those presently available. After a regulatory change that allowed for substantially increased acoustic outputs, modern DUS devices were mandated to provide the user with on-screen information (the Thermal Index, or 'TI') about ultrasound-induced temperature increments in the target tissue. The TI is generally accurate to within a factor of 2, but the factor may be as high as 6 in certain obstetric settings. Thus, informed use of and attention to the TI is strongly advised, with this admonition gaining increased emphasis if the present regulations regarding allowable acoustic outputs of DUS devices were to be further relaxed or eliminated.

Mutations induced in the HPRT gene by X-irradiation during G(1) or S: analysis of base pair alterations, small deletions, and splice errors.

Reverse transcriptase PCR was performed with mRNA obtained from HPRT mutants that had base pair alterations, or small deletions or insertions <20bp. The frequencies of mutants yielding RT-PCR products (mRNA) were the same when human EJ30 cells were irradiated in G(1) or S (3-4-fold higher for 6 than 3Gy). However, the frequencies of mutants that did not yield RT-PCR products were approximately 10-fold higher in the cells irradiated in G(1) than in those irradiated in S. Sequence analysis of RT-PCR products and genomic DNA showed that 40% of the RT-PCR products had splice errors (one or more exons not spliced into mRNA), with 64% of them due to 1-17bp deletions. Also, the distributions of molecular alterations in exons, acceptor sites, and donor sites for mutants having splice errors (observed in this study and reported by others) were similar to those reported for mutants not yielding RT-PCR products (isolated from Russian cosmonauts). In addition, we have found previously that large deletions which eliminated 1-9 exons were preferentially induced in G(1). Therefore, we postulate that the preferential induction of mutants not yielding mRNA is due primarily to splice errors that result from deletions preferentially induced during G(1). These splice errors would then result either in no message or a message that is rapidly degraded.

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.

Evidence that most radiation-induced HPRT mutants are generated directly by the initial radiation exposure.

Radiation-induced HPRT mutants are generally assumed to arise directly from DNA damage that is misrepaired within a few hours after X-irradiation. However, there is the possibility that mutations result indirectly from radiation-induced genomic instability that may occur several days after the initial radiation exposure. The protocols that commonly employ a 5-7 day expression period to allow for expression of the mutant phenotype prior to replating for selection of mutants would not be able to discriminate between mutants that occurred initially and those that arose during or after the expression period. To address this question, we performed a fluctuation analysis in which synchronous or asynchronous populations of human bladder carcinoma cells were treated with single doses of X-irradiation. For comparison, radiation was delivered during the expression period, either from an initial dose of 1.0 Gy followed by two 1.0 Gy doses separated by 24 h or from disintegrations resulting from I125dU incorporated into DNA. The mutation frequency observed at the time of replating was used to calculate the average number of mutants in the initial irradiated culture by assuming that the mutants were induced directly at the time of irradiation. Then, this average number was used to calculate the fraction of the irradiated cultures that would be predicted by a Poisson distribution to have zero mutants. There was reasonably good agreement between the predicted poisson distribution and the observed distribution for the cultures that received single doses. Moreover, as expected, when cultures were irradiated during the expression period, the fraction of the cultures having zero mutants was significantly less than that predicted by a Poisson distribution. These results indicate that most radiation-induced HPRT mutations are induced directly by the initial DNA damage, and are not the result of radiation-induced instability during the 5-7 day expression period.

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.

Molecular Biology to Radiation Oncology: A Model for Translational Research? Opportunities in basic and translational research. From a workshop sponsored by the National Cancer Institute, Radiation Research Program, January 26-28, 1997, Bethesda, Maryland.

Many exciting discoveries are being made that are providing new insights into how molecules, cells and tissues respond to ionizing radiation. There remains a need, however, to translate these findings into more effective treatments for cancer patients, including those treated with radiation therapy. This complex task will require the collaboration of scientists studying molecular, cellular and tissue responses, and those performing clinical trials of emerging therapies. The Radiation Research Program of the National Cancer Institute sponsored a workshop entitled "Molecular Biology to Radiation Oncology: A Model for Translational Research?" to bring together basic scientists and clinicians to exchange ideas and fundamental concepts and to identify opportunities for future research and collaboration. Four broad topics were addressed: signal transduction and apoptosis, the cell cycle, repair of radiation damage, and the microenvironment. The development, selection and use of appropriate experimental models is crucial to finding and developing new therapies, and opportunities exist in this area as well. This paper and the accompanying paper by Coleman and Harris that provides the viewpoint of radiation oncologists (Radiat. Res. 150, 134-147, 1998) summarize the background concepts and opportunities for translational research identified by the workshop participants.

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.

Pulsed-field gel electrophoretic migration of DNA broken by X irradiation during DNA synthesis: experimental results compared with Monte Carlo calculations.

Synchronous CHO cells were X-irradiated in G1 or mid-S phase with 30-750 Gy, and then the size distribution of DNA molecules resulting from DNA double-strand breaks (DSBs) was studied by pulsed-field gel electrophoresis (PFGE). Cells irradiated in S phase also were pulse-labeled with [3H]dThd for 15 min to compare the migration patterns of replicating DNA with those of DNA mass, measured by imaging with a CCD camera. When cells were irradiated immediately after pulse labeling, a large amount of the 3H-labeled replicating DNA was trapped in the plug, i.e. > 90% for doses < 100 Gy. As the dose increased, the percentage trapped decreased, i.e. to approximately 50% for 750 Gy. The same results were observed for DNA mass when cells were irradiated in S phase, except that much less of the DNA was trapped, i.e. approximately 60% for 70-100 Gy, which produced approximately 2-Mbp molecules, compared to approximately 10% for 750 Gy, which produced approximately 0.3-Mbp molecules. These results and the migration patterns of DNA released into the lane indicated that large molecules are trapped more readily than small molecules because they contain more replicating regions (bands with bubbles) of DNA than small molecules. Our interpretation is that as the dose increases, a greater fraction of the breaks occur between the replicating bands, thus releasing linear molecules that are not replicating. The relatively small amount of 3H-labeled replicating DNA that is released from the PFGE plug migrates aberrantly, with a small amount migrating like linear G1-phase molecules and a large amount, depending on dose, migrating much more slowly than the DNA mass from cells irradiated in G1 or S phase. To explain these results, a Monte Carlo computer program was written to introduce DSBs randomly into DNA that is configured according to a model of DNA replication that is developed in a related study (Dewey and Albright, Radiat. Res. 148, 421-434, 1997). In relating the experimental observations to the results of the Monte Carlo calculations, we assumed that (a) molecules containing replication bubbles with and without forks are trapped in the PFGE plug, (b) linear molecules and molecules with replication forks only that are < or = 8 Mbp are released into the lane, and (c) molecules having replication forks migrate more slowly than linear molecules.

Developing a model of DNA replication to be used for Monte Carlo calculations that predict the sizes and shapes of molecules resulting from DNA double-strand breaks induced by X irradiation during DNA synthesis.

A Monte Carlo computer program was written to introduce double-strand breaks (DSBs) randomly into cellular DNA that is configured according to different models of DNA replication. Then, from a review of the literature using DNA fiber autoradiography and other studies relating to rates of replication of DNA that is organized in approximately 3-Mbp regions or bands, a particular model for DNA replication was developed. Using this model, Monte Carlo calculations were made to predict the types and sizes of molecules that would result from introducing DSBs into DNA when synchronous cells are irradiated in the middle of S phase. Then results of the Monte Carlo calculations were compared with migration profiles obtained by pulsed-field gel electrophoresis (PFGE) for molecular size distributions of linear DNA molecules. For these comparisons, CHO cells irradiated in S phase also were pulse-labeled at the time of irradiation with [3H]dThd for 15 min to compare the migration patterns of 3H-labeled replicating DNA with those of the mass of S-phase DNA, measured by imaging with a CCD camera. For the Monte Carlo calculations, we assumed from the reports in the literature that molecules containing replication bubbles with and without forks would be trapped in the PFGE plug. We also assumed that those molecules that are < or = 8 Mbp, both linear and with replication forks, would be released into the lane. However, approximately 75% of the 3H-labeled DNA that is released from the plug migrated much more slowly than linear molecules, which we attributed to the slow migration of 3H-labeled molecules having replication forks not attached to bubbles. The percentages of both mass of S-phase DNA and 3H-labeled replicating DNA released from the plug, as determined by PFGE, were compared with comparable values determined from Monte Carlo calculations. A DNA replication model that provides good agreement between the PFGE results and Monte Carlo calculations is described. Furthermore, Monte Carlo methodology is presented that can be used for comparing data obtained with PFGE with results of Monte Carlo calculations that are based on different models of DNA replication and different assumptions for the migration of various types of replicating molecules.

Characterization of radiation-induced apoptosis in rodent cell lines.

For REC:myc(ch1), Rat1 and Rat1:myc(b) cells, we determined the events in the development of radiation-induced apoptosis to be in the following order: cell division followed by chromatin condensation, membrane blebbing, loss of adhesion and the uptake of vital dye. Experimental data which were obtained using 4He ions of well-defined energies and which compared the dependence of apoptosis and clonogenic survival on 4He range strongly suggested that in our cells both apoptosis and loss of clonogenic survival resulted from radiation damage to the cell nucleus. Corroboratory evidence was that BrdU incorporation sensitized these cells to radiation-induced apoptosis. Comparing the dose response for apoptosis and the clonogenic survival curves for Rat1 and Rat1:myc(b) cells, we concluded that radiation-induced apoptosis contributed to the overall radiation-induced cell inactivation as assayed by clonogenic survival, and that a modified linear-quadratic model, proposed previously, modeled such a contribution effectively. In the same context, the selective increase in radiation-induced apoptosis during late S and G2 phases reduced the relative radioresistance observed for clonogenic survival during late S and G2 phases.

Migration patterns in pulsed-field electrophoresis of DNA restriction fragments from log-phase mammalian cells after irradiation and incubation for repair.

An assay system was developed to detect changes of restriction fragment profiles obtained after pulsed-field gel electrophoresis (PFGE) for DNA from mammalian cells that were irradiated and incubated for repair. DNA was prepared from irradiated log-phase human melanoma cells (MRI 221) after incubation for repair (6 h) and was digested with the rare-cutting restriction enzyme NotI (RE) prior to PFGE separation. DNA-fragment size distributions were compared to the respective PFGE profiles from unirradiated controls. After doses of 5 and 10 Gy (plus a 6-h incubation for repair), the relative amount of DNA retained in the plug during PFGE was increased. For higher doses (30 and 60 Gy), this phenomenon was superimposed by the residual fragmentation (25-30% of the initial breakage of 0.42 dsb/100 Mbp/Gy). Since irradiated cells accumulated in S phase during incubation for repair, a correction for the reduced electrophoretic migration of DNA from S-phase cells was necessary, and a 0.5% increase in DNA retention per 1% S-phase increment was found. However, the % retention for the 5 Gy plus repair sample was significantly higher than for the S-phase adjusted control (p < 0.01). Radiation induced DNA-protein crosslinks cannot account for the observed phenomenon because of the extensive proteolysis in DNA preparation, and also a loss of restriction enzyme recognition sites appear to be an unlikely explanation from simple quantitative considerations. Based on the recent observation of misrejoining during dsb repair, it is proposed that the incorrect joining of DNA ends also causes a more random distribution of replicating DNA in restriction fragments derived from cells in S phase after incubation for repair. This process would necessarily increase the proportion of DNA unable to migrate in PFGE.

Apoptosis induced by X-irradiation of rec-myc cells is postmitotic and not predicted by the time after irradiation or behavior of sister cells.

Rat embryo cells expressing the c-myc oncogene (rec-myc) were studied by time-lapse microscopy to determine whether radiation-induced apoptosis occurred before or after mitosis. Following X-irradiation with 9.5 Gy, cells were imaged every 3 min for 6 days. Episodes of apoptotic blebbing were very consistent from cell to cell, lasting 30-60 min, followed by cessation of movement and cell death. In contrast, the time of initiation of apoptotic blebbing was unpredictable. At least 96% of the apoptotic episodes were postmitotic, after one to four cell divisions and 2-97 h after a given division. Sister cells often behaved differently from one another, with apoptosis in one sister occurring many h or several divisions after apoptosis in the other. Thus, the onset of radiation-induced apoptosis in rec-myc cells is not strictly programmed but may result from the segregation of chromosome aberrations in the postirradiation generations.

p53-dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma.

PURPOSE: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.