Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80: analysis by cDNA microarray.

The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes.

Butachlor, a suspected carcinogen, alters growth and transformation characteristics of mouse liver cells.

Butachlor is a widely used herbicide in Asia and South America. Previous investigations have indicated that it is a suspected carcinogen. To understand more about the biological effects of butachlor on cultured cells and the mechanism(s) of its carcinogenicity, we studied the alteration of the growth characteristics that was induced by butachlor in normal mouse liver cells (BNL CL2). This study demonstrates that butachlor decreases the population-doubling time of BNL CL2 cells, suggesting that it stimulates cell proliferation. To support this finding, a thymidine incorporation assay was conducted and a similar result that butachlor stimulates cell proliferation was elucidated. In addition, we show that butachlor increases the saturation density of the BNL CL2 cells. When combined with the tumor initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), butachlor transforms cells efficiently, as demonstrated by loss of contact inhibition. These findings indicate that butachlor alters the growth characteristics of BNL CL2 cells and suggest that butachlor may induce malignant transformation through stimulation of cell proliferation, alteration of cell cycle regulation, and suppression of cell density-dependent inhibition of proliferation.

Chromosomal damage in long-term residents of houses contaminated with cobalt-60.

Chromosomal translocations in people who have lived in houses contaminated with radiation were substantially raised compared with controls. Retrospective biological dosimetry indicated cumulative exposures less than 1.0 Gy, which were lower than values derived from physical measurements.

Effects of gamma knife radiosurgery for brain tumors: clinical evaluation.

BACKGROUND: Gamma knife radiosurgery is a safe and effective alternative to microsurgery in the management of selected intracranial lesions. In our initial three-year experience with gamma knife radiosurgery, 431 patients were treated using this method. This report presents the treatment results for three different types of brain tumors: benign meningiomas, malignant metastases and gliomas. METHODS: A retrospective study was performed to analyze a consecutive series of 71 meningiomas, 31 metastatic tumors and 21 gliomas treated by gamma knife radiosurgery between March 1993 and May 1996. The treatment results were investigated using regular magnetic resonance examinations and tumor volume measurement at six-month intervals to observe sequential changes of the tumors. Patients with meningiomas were further divided into three groups according to the peripheral radiation doses: high-dose (20-17 Gy, n = 18), medium-dose (16-15 Gy, n = 33) and low-dose (14-12 Gy, n = 20). The Generalized Estimation Equation was applied to compare treatment results in these three groups with different doses and tumor volumes. RESULTS: Volume measurements of the 71 meningiomas showed that 76% decreased in size, 16% stabilized and 8% increased in size. The volumes increased most frequently in the early stage (6-12 months) after treatment and subsequently regressed after the twelfth month. The tumor control rate for meningiomas in our three-year follow-up was over 90%. For meningiomas, the statistical analysis showed that both the radiation dose and tumor volume were significantly related to the development of adverse radiation effects (p < 0.05). In metastatic tumors, rapid tumor regression after radiosurgery was found in 87% of the patients. In gliomas, radiosurgery effectively inhibited tumor growth in selected patients with small, circumscribed, less infiltrative tumors. Ependymomas and low-grade astrocytomas had more favorable outcomes than other gliomas. CONCLUSIONS: Gamma knife radiosurgery is effective for controlling tumor growth in benign meningiomas for up to three years after surgery. In selected cases of malignant metastasis and gliomas, most patients appeared to benefit from the treatment with symptomatic improvement and prolonged survival. Treatment strategy and dose selection in radiosurgery should be adjusted to optimize tumor control and avoid adverse radiation effects.

Comparison of the effects of hydralazine on tumor and normal tissue blood perfusion by MRI.

PURPOSE: The differential effects on blood perfusion of the vasodilator hydralazine (HYD) between tumor and normal muscle have been measured using the dynamic enhanced-magnetic resonance imaging (DE-MRI) technique. METHODS AND MATERIALS: DE-MRI is a noninvasive method of determining blood perfusion in tumors and normal tissues using the MR contrast agent Gd-DTPA. Hydralazine is currently being used in an attempt to increase tumor response to bioreductive agents and to hyperthermia. RESULTS: We show that a dose of 1.2 mg/kg HYD causes an increase in tumor perfusion while doses > or = 2.5 mg/kg cause a decrease in tumor perfusion. The latter was accompanied by a dose-dependent increase in normal muscle perfusion consistent with the "steal effect." CONCLUSION: This study demonstrates the sensitivity of the DE-MRI technique and its capability of providing estimates of blood perfusion in normal and tumor tissue as well as in smaller regions of a solid tumor. Such features would make it clinically useful in the study of tumor response to radiation therapy and chemotherapy in patients.

Determination of changes in tumor blood perfusion after hydralazine treatment by dynamic paramagnetic-enhanced magnetic resonance imaging.

Magnetic resonance imaging, using the paramagnetic chelate gadopentetate dimeglumine as a perfusing agent, was used to investigate the effect of the vasoactive drug hydralazine on tumor blood perfusion. The method requires measurements of the magnetic resonance image intensity changes with time on a pre-selected region of interest in the tumor image, immediately following intravenous injection of gadopentetate dimeglumine. The present study showed that the initial slope of the intensity-time curve can be used, to a first approximation, to infer tumor blood perfusion. With the dynamic imaging technique, it was demonstrated that, in the KHT sarcoma implanted intramuscularly in the hind leg of C3H/HeN mice, intraperitoneal administration of hydralazine reduced the volume-averaged tumor blood perfusion in a dose-dependent manner. The intrinsically high spatial resolution of magnetic resonance imaging allows a detailed study of the heterogeneous nature of tumor blood perfusion. The potential applications of this imaging technique to study the differential effects of hydralazine on perfusion between tumor and normal tissues will be discussed. The clinical utility of the technique should be promising because of its non-invasive nature.

The dependence of proton longitudinal and transverse relaxation times on cell-cycle phase: mouse MCA-transformed 10T1/2 TCL-15 cells.

Attempts to determine proton NMR longitudinal relaxation times (T1) as a function of cell-cycle stage using cells synchronized by chemical methods have yielded conflicting results (P. T. Beall, C. F. Hazlewood, and P. N. Rao, Science 192, 904 (1976); R. N. Muller et al., FEBS Lett. 114, 231 (1980); D. N. Wheatley, et al., J. Cell Sci. 88, 13 (1987]. This has raised the question whether a true dependence of T1 on cell-cycle phase exists. In the present study, the centrifugal elutriation technique was used to obtain relatively pure, synchronized cell populations of TCL-15 cells (a methylcholanthrene-transformed line of mouse 10T1/2 cells) for measurement of proton NMR relaxation rates. This technique provides a means to procure synchronized cell populations without the use of chemical agents as in the above-cited investigations and therefore avoid possible effects caused by the chemical agents of the NMR relaxation processes. Both T1 and the transverse relaxation time, T2, of water protons in synchronized-cell pellets obtained in this study, exhibited a dependence on cell-cycle phase at least for the first half of the cell cycle (G1 to S). Cells in G1 phase exhibited quantitatively higher T1 and T2 relaxation times compared to those measured for cells in mid S phase. Such changes were found to correlate with changes in water content. The distribution of cell-cycle phases of each cell population was determined by the DNA histogram using flow cytometric methods. Possible relaxation mechanisms which may contribute to the cell-cycle-specific phenomena of the intracellular T1 and T2 times are discussed.

Basic radiobiological investigations of fast neutrons.

The radiobiological properties of a cyclotron-produced 43-MeV (p----Be) fast-neutron beam relative to gamma rays have been investigated using Chinese hamster V79 cells in culture. As expected, the relative biological effectiveness (RBE) of this neutron beam for cell killing was shown to increase as dose decreased, and the effectiveness per unit dose was slightly less compared to a 25-MeV (d----Be) neutron beam. By tracing single cells that formed microcolonies after irradiation, we found cell proliferation kinetics to be retarded to a greater extent by fast neutrons than by gamma irradiation. Following either neutron or gamma irradiation, a fraction of the irradiated cells failed to divide in the first postirradiation division and another fraction could produce as many as four generations of progeny before proliferation stopped. The properties of these cells presumed to be destined for death suggest that more than one mechanism and/or multistep process underlies the radiation-induced proliferative death. The fast-neutron beam was also found to be more effective quantitatively than gamma rays in producing DNA double-strand breaks (DSBs, measured by nondenaturing filter elution), and G1-phase chromosome fragments (measured by the premature chromosome condensation technique). However, the reverse was observed for DNA single-strand breaks (SSBs, measured by alkaline filter elution or hydroxylapatite uncoiling). Interestingly, both fast neutrons and gamma rays produced a large component of SSBs and DSBs with a fast-rejoining time constant of about 2-5 min, which appears to be independent of dose. The latter results could not resolve the possibility of lengthening the repair-time constant by increasing radiation dose within the range that is reflected by the shoulder of the survival curve, and consequently did not support the idea of repair saturation as a mechanism for the presence of the shoulder. The RBE for the hypoxanthine phosphoribosyl transferase mutation frequency per survivor at the 10% survival level was estimated to be 2.5, a value that is comparable to the RBE (2.1) for cell killing at the same survival level. Although most of the above-mentioned findings are compatible qualitatively with the relatively high-LET (linear energy transfer) nature associated with the fast-neutron beam, the significance of the action attributable to the mixture of LET could not be delineated in these experiments. Further, the biological significance of DSBs and chromosome aberration and the molecular mechanisms responsible for the repair and expression of these damaging processes remain to be elucidated.

The response of the KHT sarcoma to radiotherapy as measured by water proton NMR relaxation times: relationships with tumor volume and water content.

The potential application of magnetic resonance imaging (MRI) to predict tumor response to radiotherapy is investigated. The water proton spin-lattice and spin-spin relaxation times (T2 and T2, respectively) of murine sarcomas (designated KHT) were measured shortly after excision. This study has demonstrated significantly different responses in T1 and T2 between the control and the irradiated tumors at various times following single doses of X rays. Quite generally, the changes in relaxation times correlated with the changes in tumor water content, indicating that the MR relaxation-time probes are fairly sensitive to radiation-induced edema and dehydration. The possible relationships between the T1 and T2 responses and radiobiological effects such as those on tumor blood flow, vascular permeability, physiological state of cells, and cell death are discussed. It is conceivable that the findings obtained from this investigation could be extended to in situ studies for potential applications in clinical radiotherapy.

Therapeutic gain factors for fractionated radiation treatment of spontaneous murine tumors using fast neutrons, photons plus O2(1) or 3 ATA, or photons plus misonidazole.

Therapeutic gain factors (TGFs) have been determined for three spontaneous tumors of the C3H mouse treated by photons + normobaric oxygen (O2(1) ATA), photons + hyperbaric oxygen (O2 3 ATA), photons + misonidazole, or fast neutrons. The tumors were early generation isotransplants of spontaneous tumors: MCaIV, a mammary carcinoma; FSaII, a fibrosarcoma; and SCCVII, a squamous cell carcinoma. The tumors, transplanted to the right leg, were 6 mm at start of treatment. Normal tissue responses studied were acute reaction of normal skin (all treatment modalities) and LD50 following irradiation of the upper abdomen (in test of photons + O2 at 1 or 3 ATA). Thus both the tumor and normal tissues would be classified as "acute responding." All subject tissues were at congruent to 34.5-35 degrees C at irradiation. Treatments were based on d(25)Be or p(43)Be fast neutron beams, 60Co and 137Cs photon beams. Treatments were given in 5 or 15 equal doses in 5 days. For photon treatments, TGFs (air/O2 3 ATA) were substantially and significantly larger than 1 for all three tumor systems treated at small or large doses per fraction when related to skin or abdominal tissue responses. The TGFs (air/O2 1 ATA) were greater than 1 at small doses per fraction for MCaIV and FSaII for skin as the normal tissue; the TGFs for all three tumors and at all doses per fraction would be greater than 1 when related to upper abdominal tissues. TGFs (O2 1 ATA/O2 3 ATA) for photon irradiation greater than 1 were found only for SCCVII and that obtained for both large and small doses per fraction. Misonidazole achieved impressive TGFs (air/air + miso or air/O2 1 ATA + miso); the drug was tested only at 10-12 Gy/fraction and relative to skin. RBEs(FN) for the three tumors were lower at 1.5-2 Gy(FN)/fraction than at 5-6 Gy(FN)/fraction, i.e. the opposite to that reported for normal tissue (RBE increases with decreasing dose per fraction). A TGF (relative to skin reaction) greater than 1 for fast neutron therapy was found only for SCCVII when treated at large doses/fraction; this was true for air or O2 1 ATA conditions.

Sequential exposures of mammalian cells to low- and high-LET radiations. II. As a function of cell-cycle stages.

The synergistic effects of low- and high-LET radiations were further studied with partially synchronized Chinese hamster V79 cells. Principally, nearly monoenergetic 425 MeV/u neon ions and 570 MeV/u argon ions produced near the Bragg peak were employed as the high-LET radiations and 225 kVp X rays as the low-LET counterpart. It was found that the killing effect due to damage interaction after sequential irradiations with the particle beam and X rays varies throughout the cell cycle. The greatest effect was observed in late-S phase which was most resistant to either of the radiations. The effect was quantitatively less in the G1/S border and in G2. Effects on pure mitotic cells have not been investigated in this study. For all cell stages studied, a dose of high-LET particles modified the shape of the X-ray survival curve in a way similar to the modification predicted by an appropriately selected X-ray dose. This finding suggests that the mechanism for the synergistic effects is similar to that operating for sequential treatments with X rays alone. Experiments with an S population, either incubated at 37 degrees C or room temperature between fractionation of high- and low-LET radiation treatments further verified that the damage involved is a repairable type. At a certain fractionation interval (6 to 8 h) following a dose of high-LET treatment, initially asynchronous cells were found to be very sensitive to X-irradiation. It is noteworthy that the net killing measured at this "radiosensitive window" was as effective as the killing observed by "immediately" sequential treatments with the same doses of high- and low-LET radiations. Such a time window also existed when the order of the treatment sequence was reversed except that the time of occurrence was earlier and the window was broader. This sensitization effect may be explained by radiation-induced G2 arrest together with an increase of radiosensitivity as the previously irradiated cells progress into S phase. Radiotherapy strategies using combined high-LET and low-LET radiations for rapidly proliferative tumors are presented.

Survival responses and potentially lethal damage repair of normal 10T1/2 and its transformed TCL 15 cells after irradiation with 43 MeV proton produced neutrons.

Normal 10T1/2 fibroblasts and their transformed counterparts (TCL 15) were used in order to evaluate the RBE, TGF and PLD repair for a therapeutic fast neutron beam (43 MeV proton----Be). For plateau-phase culture, the RBE of 10T1/2 cells were 2.0 and 1.7 at 10 and 1% survival levels respectively and 1.3 from D0. The corresponding RBE values of TCL 15 cells were 2.1, 1.8 and 1.5, and thus the TGF values were 1.05 and 1.1 respectively at 10% and 1% survival levels and 1.8 from D0. For log-phase culture, the survival responses of 10T1/2 and TCL 15 cells to 60Co gamma-rays or neutrons were not significantly different (the RBE at 10% was 2), and thus the TGF value was unity. For neutrons, as a rule no PLD repair has been reported in vitro and in vivo in previous studies. However, in the present study PLD repair occurred in plateau 10T1/2 and TCL 15 cells irradiated with neutrons.

The effects of a static magnetic field on DNA synthesis and survival of mammalian cells irradiated with fast neutrons.

The effects of a static magnetic field (0.75 T) on DNA synthesis and survival were examined with Chinese hamster V79 cells in cultures with and without fast-neutron irradiation. We found that the magnetic field applied alone for up to several hours did not cause a significant effect in either the rate of DNA synthesis or cell viability; the latter was assayed by colony formation. When cells were exposed simultaneously to the magnetic field and fast neutrons, the effects resembled those observed with neutrons alone. This was the case for both inhibition of DNA synthesis and cell killing. Cells irradiated first with neutrons followed immediately by 1 h of magnetic field exposure showed a dose-survival response curve indistinguishable from that of neutrons alone. These data suggest that the biological effect due to the magnetic field is negligible and that the presence of the magnetic field either during or subsequent to fast-neutron irradiation does not affect the neutron-induced radiation damage or its repair.

Possible importance of PLD repair in the modulation of BrdUrd and IdUrd-mediated radiosensitization in plateau-phase C3H10T1/2 mouse embryo cells.

C3H10T1/2 mouse embryo cells exhibiting strong contact inhibition of growth at confluency were grown in the presence of 5-bromodeoxyuridine (BrdUrd) or 5-iododeoxyuridine (IdUrd) (0-1.2 microM) with daily refeeding and exposed to gamma-rays (6 Gy) either in the logarithmic or the plateau phase of growth. Sensitization to radiation was observed in both growth states with increasing concentration of BrdUrd or IdUrd but the degree of sensitization achieved was lower for plateau-phase cells. Because the degree of [H3]BrdUrd incorporation was found to be similar in exponentially growing and plateau-phase cells, it is hypothesized that the radiosensitization caused by pyrimidine analogues may be affected by the physiological state of the cells at the time of irradiation. Delayed plating of plateau-phase cells (6 h) caused an increase in survival, indicating repair of potentially lethal damage (PLD). A greater increase in cell survival was observed in cells that had been grown in the presence of BrdUrd and IdUrd and it was found to increase with increasing concentrations. This analogue-concentration dependent PLD repair activity resulted in an almost complete loss of the radiosensitizing effect in delayed plated plateau-phase cells up to a concentration of about 0.6 microM of BrdUrd and IdUrd. Both compounds, but especially BrdUrd, caused a relaxation in the mechanism of contact inhibition and led to higher cell densities in the plateau phase. The results suggest that repair and/or expression of PLD might be involved in the mechanism underlying BrdUrd and IdUrd-mediated radiosensitization and point out the potential importance of PLD repair in the modulation of the radiosensitizing effect of these compounds in their clinical application.

Repair and fixation of potentially lethal damage (PLD) as demonstrated by delayed plating or incubation with araA in contact inhibited refed plateau-phase C3H mouse embryo 10 T1/2 cells grown in the presence of BrdUrd.

C3H mouse 10 T1/2 cells showing strong inhibition of growth at confluency were grown under daily refeeding in the presence of BrdUrd (from 0 to 1 microM) and exposed to gamma-rays either while exponentially growing or in the plateau phase. An increase in radiosensitivity was observed in both growth conditions mainly reflected by a reduction in Dq. Greater radiosensitization was observed in exponentially growing than in plateau-phase cells, and 3-4 times higher BrdUrd concentrations were required in plateau-phase cells for similar potentiation in killing. This effect could not be entirely attributed to a reduction in BrdUrd incorporation since measurements with 3H-BrdUrd showed reductions in incorporation between only 17-47% in plateau-phase cells. The rate of repair of potentially lethal damage (PLD) as demonstrated by delayed plating was not affected by the incorporation of BrdUrd, but the amount of repair (measured as the relative increase in cell survival) was higher for BrdUrd-containing cells. Post-irradiation treatment of cells in the plateau-phase (no BrdUrd) with 9-beta-D-arabinofuranosyladenine (araA) caused fixation of radiation-induced PLD. AraA treatment of cells grown in the presence of various amounts of BrdUrd also caused fixation of PLD, but resulted in survival levels similar to those observed with cells growing in BrdUrd-free medium. This result indicates that BrdUrd mediated radiosensitization cannot be observed when cells are prevented from repairing PLD by postirradiation incubation with araA. Based on these findings we propose that the mechanism of radiosensitization by BrdUrd incorporation might be, by increasing probability of fixation, mediated by the postirradiation progression of cells through the cycle, of a sector of PLD also sensitive to post-irradiation treatment with araA. For this sector of PLD the term alpha-PLD has been proposed.

Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms.

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.

Effect of induced field inhomogeneity on transverse proton NMR relaxation in tissue water and model systems.

The effect of induced field inhomogeneity (IFI) on transverse NMR relaxation of water protons in tissue has been investigated by examining the field dependence of the effective transverse relaxation rates (1/T2 eff) for in vitro canine brain tissue samples. At fields of 0.47, 2.35, 7.05 T (corresponding to 20, 100, and 300 MHz, respectively) the transverse relaxation rates for both white and gray matter samples follow a field dependence of the form 1/T2 eff = C0 + C1 B0, where B0 is the applied field. The linearly dependent term, C1 B0, which reflects the IFI contribution, does not contribute much (i.e., less than 20%) at fields less than 2.0 T. However, at greater field strengths the contribution is appreciable, e.g., greater than 60% at 7.0 T. Results from model systems of glass beads are also reported to illustrate IFI effects. For both the model systems and canine brain tissue samples, the effects of restricted diffusion are qualitatively evident in Hahn spin-echo experiments.

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays.

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.