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Background Rodent studies show that neurogenesis is necessary for mediating the salutary effects of antidepressants. Nonhuman primate (NHP) studies may bridge important rodent findings to the clinical realm since NHP-depression shares significant homology with human depression and kinetics of primate neurogenesis differ from those in rodents. After demonstrating that antidepressants can stimulate neurogenesis in NHPs, our present study examines whether neurogenesis is required for antidepressant efficacy in NHPs. Materials/Methodology Adult female bonnets were randomized to three social pens (N = 6 each). Pen-1 subjects were exposed to control-conditions for 15 weeks with half receiving the antidepressant fluoxetine and the rest receiving saline-placebo. Pen-2 subjects were exposed to 15 weeks of separation-stress with half receiving fluoxetine and half receiving placebo. Pen-3 subjects 2 weeks of irradiation (N = 4) or sham-irradiation (N = 2) and then exposed to 15 weeks of stress and fluoxetine. Dependent measures were weekly behavioral observations and postmortem neurogenesis levels. Results Exposing NHPs to repeated separation stress resulted in depression-like behaviors (anhedonia and subordinance) accompanied by reduced hippocampal neurogenesis. Treatment with fluoxetine stimulated neurogenesis and prevented the emergence of depression-like behaviors. Ablation of neurogenesis with irradiation abolished the therapeutic effects of fluoxetine. Non-stressed controls had normative behaviors although the fluoxetine-treated controls had higher neurogenesis rates. Across all groups, depression-like behaviors were associated with decreased rates of neurogenesis but this inverse correlation was only significant for new neurons in the anterior dentate gyrus that were at the threshold of completing maturation. Conclusion We provide evidence that induction of neurogenesis is integral to the therapeutic effects of fluoxetine in NHPs. Given the similarity between monkeys and humans, hippocampal neurogenesis likely plays a similar role in the treatment of clinical depression. Future studies will examine several outstanding questions such as whether neuro-suppression is sufficient for producing depression and whether therapeutic neuroplastic effects of fluoxetine are specific to antidepressants.

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to β-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

We examined, by the fluorescent halo assay, alterations in the nucleoid structure (structure formed from cells under mild lysis conditions: in non-ionic detergent Triton X-100, 0.0005% and 1.5 mol/l NaCl) of L5178Y (LY) cell sublines which had been untreated, treated with reducing/chelating agents (beta-mercaptoethanol or sodium diethyl dithiocarbamate (DDTC(Na))) or X-irradiated. These sublines differ in radiation sensitivity: LY-R is more resistant (D0 = 1.1 Gy) and LY-S more sensitive (D0 = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI) (0.5 to 50 micrograms/ml) at pH 6.9 or 9. The maximal DNA unwinding in PI was obtained at 7.5 micrograms/ml PI, at both pH 6.9 and 9 in both sublines; the maximal halo diameter was larger in LY-S than in LY-R cells. In nucleoids from both sublines DNA could be rewound at higher (10-50 micrograms/ml) PI concentrations both at pH 6.9 and 9. This ability was impaired by mercaptoethanol or DDTC(Na) (at pH 9) or by X-irradiation, indicating damage and/or alteration in the DNA superhelical structure. The susceptibility to reducing/chelating agents was greater in LY-S than in LY-R nucleoids, pointing to differences in chromatin structure between these sublines. The amount of X-ray-inflicted damage was higher, when measured at pH 9 than at pH 6.9 and was about twice larger in LY-S than in LY-R nucleoids, when the cells were irradiated with the same X-ray dose.(ABSTRACT TRUNCATED AT 250 WORDS)

A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillary melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.

We have measured survival after successive 2-Gy doses of X rays in HeLa cells and 1-Gy doses in cells of the nonimmortalized human fibroblast cell line AG15-22 under conditions where any effect of cell proliferation during multifraction X irradiation has been factored out. When HeLa cells in parallel series of (pseudo)hybrid spheroids (i.e. in agglomerates consisting of a mixture of supralethally irradiated HeLa feeder and viable HeLa cells) were exposed to n daily radiation doses and then trypsinized and exposed to the last dose, the surviving fraction at 2 Gy (SF2) declined exponentially from 0.55 +/- 0.01 to 0.31 +/- 0.01 after the fifth fraction. In monolayer HeLa cell cultures, the decline in SF2 was smaller but significant and was not influenced by the presence of feeder cells. Pure spheroids, composed entirely of viable HeLa cells, showed the same decline in SF2 as did monolayer cells. The cumulative-effect curve (i.e. the product of SF2 values) was linear-quadratic with the quadratic term increasing in the order monolayer, pure spheroids, pseudohybrid spheroids. SF2n and D0Eff (deduced from the initial SF2) severely underestimated cumulative radiosensitivity. This cumulative effect is clearly associated with the proximity of lethally irradiated cells and might be explained by differential population shifts in the course of the multifraction regimen. Similarly, AG15-22 cells irradiated with daily 1-Gy doses of X rays showed a larger increase in radiosensitivity when in hybrid spheroids than when in pure spheroids. However, for the AG15-22 cells, SF1 was twofold lower for the former than for the latter condition and remained constant for both conditions rather than decreasing with increasing fraction number. This large radiosensitizing effect remains unexplained.

Modulation of radiosensitivity by components of the extracellular matrix (ECM) and cytoskeletal elements has not been adequately studied. Although differences in the radiosensitivities of cells grown as monolayers, as spheroids, or grown in vitro in animal models are known, explanations have in the past neglected possible influences by the ECM and cytoskeleton. Using collagen gel cultures, it is shown that the fibrillar component of the ECM (which is responsible for cell anchorage) induces shifts in radiosensitivity. The effect is critically dependent on the affinity of the cell type towards collagen. The shifts in radiosensitivity induced by ECM alteration are manifested as changed Dq values. By applying four specific cytoskeletal poisons which either stabilize or destabilize specific cytoskeletal elements, the involvement of microfilaments and microtubuli was qualitatively appraised. Cytochalasin B, which destabilizes microfilaments (by preventing polymerization), caused a significant rise in radioresistance. This rise was due to increased D0. Although the cellular morphological change accompanying cytochalasin B treatment was essentially similar to that obtained with trypsin, the respective shifts in radioresponses were qualitatively different and opposite, suggesting differences in mechanism of action.

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H2O2, the effect being more pronounced at 37 degrees C than 0 degree C. Initial DNA damage after H2O2 treatment (both temperatures, 5 min) has been estimated by the 'comet' assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at 0 degree C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H2O2 treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H2O2 and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H2O2 and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.

Publisher Summary This chapter discusses radiation induced damage in chromosomal DNA molecules. A chromosome structure that contains either circular or parallel DNA molecules introduces serious problems in traditional interpretations of genetic structure and function. Strand breakage has usually been determined by measuring the sedimentation rates for irradiated and unirradiated DNAs, deducing their number average molecular weights. For DNA molecules up to 2–5 X 108 Da, reasonable estimates of the average number of breaks induced or rejoined per molecule can be obtained. However, molecules of this size are much smaller than the DNA content of the average mammalian chromosome. The measurement of single-stranded breaks (SSB) under neutral conditions avoids the complications introduced by alkaline conditions and provides useful estimates of radiation yield. DNA double-stranded breaks (DSB) are measured by sedimentation and gel electrophoresis techniques similar to those for SSB. However, for DSB measurements, nominally neutral pH conditions are employed. Conventional gel electrophoresis has been used mainly for the measurement of DSB in DNA restriction fragments and in small viral/bacteriophage DNAs. Radiation may induce DNA damage nonrandomly in the lateral extensions of the chromatid backbone. The assumed close alignment of four segments of the circular DNA duplex molecule in the lateral extensions, and a possible close association with lipoprotein structures, implies that traversals of a local region by ionizing particles are more likely to induce multiple damage sites within short regions of the DNA molecule than would be expected for random dispersions of the DNA.

Our data (Reddy et al., Radiat. Res. 141, 252-258, 1995) on the kinetics of the repair of potentially lethal damage in log-phase Chinese hamster V79 cells are used to test some predictions which arise from the different assumptions of the repair-misrepair (RMR) (C. A. Tobias, Radiat. Res. 104, S77-S95, 1985), lethal-potentially lethal (LPL) (S. B. Curtis, Radiat. Res. 106, 252-270, 1986) and double-strand break (DSB) (J. Y. Ostashevsky, Radiat. Res. 118, 437-466, 1989) models. The LPL model defines the time available for repair of PLD ( $t_{{\rm rep}}$ ) as the time taken to reach maximal survival in a delayed-plating recovery experiment. Those data show that after this time has elapsed, contrary to the expectation of the LPL model, survival can be increased by changing the medium used for delayed plating from fresh growth medium to conditioned medium. According to the RMR model, all potentially lethal lesions should also be committed by that time and be unavailable for repair in the ne...

Previous observations have shown that cells cultured in standard growth medium (100%) demonstrated similarly enhanced survival when incubated postirradiation either in non-growth-promoting conditioned medium or in growth-promoting 40% growth medium (Reddy and Lange, Radiat, Res. 119, 338-347, 1989). From these results, it was suggested that nutrient dilution altered radiosensitivity by a mechanism independent of progression of cells through the cell cycle. In this study, we have examined the effects on radiosensitivity of incubation in 40% growth medium prior to irradiation on both log- and plateau-phase Chinese hamster V79 cells and the effects on the distribution of cells in the cell cycle of incubation in 40% or 100% growth medium before and after irradiation. Radioresistance increased by a factor of 1.5-1.6 compared to 100% growth medium for both log-phase and plateau-phase cells cultured in 40% growth medium prior to X irradiation and incubated in either 40% growth medium or conditioned medium after X irradiation. The cell cycle distributions of log-phase cells in 100% and 40% growth medium before irradiation were identical. The change in cell cycle distribution induced by 10 Gy did not differ among log-phase cells incubated for 3 h postirradiation in 100% growth medium, 40% growth medium or conditioned medium. These results, in addition to supporting our previous conclusions, demonstrate that culturing prior to irradiation in 40% growth medium alone increases cell survival and that incubation in 40% growth medium before and after irradiation maximizes the survival of V79 cells.

The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth mediummore » but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation. 21 refs., 4 figs., 1 tab.« less

We have read the paper by Olive and MacPhail (1) with great interest. Their data indicate that: (a) "trypsin did not influence cell killing by ionizing radiation" (i.e., the radiation sensitivity of round cells in spheroids with and without trypsin treatment was the same), and (b) the increase in the radiation resistance and the limited DNA unwinding ability (as measured by the alkaline unwinding assay) of V79 spheroid cells appear to be contingent upon a change in cell shape. Their results and their interpretation of our data are pertinent to the observations on the relationship between radiosensitivity, trypsin, cell shape, and chromatin structure published by us for V79 (S-171) cells (2-5). Our data show that: (a) trypsin can radiosensitize spread monolayer cells (2-5); (b) trypsin cannot radiosensitize round mitotic cells (2), round cells in spheroids (4), or round mouse lymphoma cells (L5178Y-S) in suspension (5); (c) radiosensitization by trypsin is related to cell shapedependent chromatin structure (2-5); and (d) the degree of unwinding ofchromatin in round cells is higher than that of spread monolayer cells (5). There has also been sporadic mention of a possible radiosensitizing effect by trypsin [(6, 7), and see Ref. (2) for additional references on trypsin]. The observation by Olive and MacPhail (1) that trypsin did not radiosensitize round cells in spheroids is essentially the same as our results for round mitotic cells (2) or round cells in spheroids (4) or for round L5178Y-S cells (5). However, their other observations that spheroid cells were more radioresistant than monolayer cells and that the degree of DNA unwinding in the former cells was lower than that of the latter cells are not in agreement with our results. Our results showed that the radiosensitivity and the degree of DNA unwinding (as measured by the nucleoid halo technique) of monolayer and spheroid cells treated with trypsin was nearly the same (5). The reason for this difference in results is not clear. Lest any confusion arise from their misquotation of our observations (2-5) in their paper (1) pertaining to the effects of trypsin on radiosensitivity, we note the following: In the last sentence of the first paragraph of the Discussion, they state: "Recent results suggest that treatment of V79-171S cells with trypsin increases their resistance to radiation damage, an effect which appears to be related to differences in cell shape between monolayers and spheroids (18)". [Their Ref. (18) is Ref. (4) here]. What we have repeatedly shown is that: (a) spread cells in monolayers treated with trypsin either immediately before or immediately after irradiation are more radiosensitive than those cells which have been treated with trypsin 2-3 h after irradiation and repair incubation, and (b) the radiosensitization under immediate plating conditions is correlated with the rounding of spread cells by trypsin (2-5). Reference to mouse lymphoma cells (L5178Y) as monolayers in Table I appears to be a typographical error.

Evidence is presented for the interaction of X irradiation, slightly toxic levels of chloroquine, and mild hyperthermia in the inactivation of colony-forming ability in asynchronous HeLa cells. A three-way interaction was observed which resulted in the potentiation of radiation-induced lethality. There was little evidence of toxicity in unirradiated cells incubated for 3 h with 0.1 mM chloroquine at either 37 or 41 degrees C. The radiopotentiation factor, which is similar to the dose modification factor, was determined from dose-response curves by relating the reciprocal of the slope (D0) of the reference survival curve to that of the survival curve of cells receiving the combined postirradiation treatment with chloroquine and mild hyperthermia. Radiopotentiation factors larger than 1.7 were obtained irrespective of whether the reference D0's were obtained from survival curves for cells irradiated at 37 degrees C without drug or from cells receiving postirradiation treatment with heat or drug only.

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.

The semiautomated sucrose gradient system of Lange and Liberman (1974) (Anal. Biochem.59, 129–145) has been used to determine an accurate value of the exponent k of the Burgi and Hershey equation (1963) (Biophys. J. 3, 309–321) under conditions of 1M salt. A complete analysis of the variance of k was done for data from both systems. The results lead to the conclusion that the value k = 0.401 (±0.012 for reproducibility; ±0.019 for total error) obtained here is considerably more accurate than that of Burgi and Hershey (even when corrected for DNA concentration effects and the improved size estimates of T4 DNA size). The use of the Burgi and Hershey relationship and a T4 DNA metric to determine molecular weights of mammalian DNA requires the utmost precision in the exponent if useful estimates are to be obtained. This also has implications for double-strand breakage and repair estimates.

The substance which inhibits brain formation in the regenerating planarian Dugesia etrusca was found to be a large molecule, at least in part protein, which electrophoreses as an electronegative moiety in pH 6.8 buffer. A model is presented, based on this finding and previous studies, which proposes an electrochemical mechanism for the control of polarity and possibly for the maintenance of tissue organization in planarians. It is proposed that a bioelectric field exists and moves the electronegative brain-inhibiting substance in a posterior direction, establishing polarity. This model explains the polarity reversal experiments using external fields and many of the previously unexplained classical planarian experiments. Data are presented demonstrating the existence, magnitude, and polarity of this bioelectric field, which is not greatly altered upon decapitation, all in accord with predictions of the model.

The Stationary or Plateau-Phase of commonly used rodent cell lines like the V79 are often assumed to be quiescent (non-mitotic). An analysis of cell turnover in V79 plateau-phase cultures through BrUdR-incorporation combined with FUdR-block and light exposure (S-phase cytocide) revealed such cultures to be in a state of kinetic equilibrium. Even when the state of maximal permissible density was acquired, at least 50% of the population of cells were cycling within the time for one population doubling. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turnover-rate was reduced. Our assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing and confluent cultures. Elimination of cycling cells by S-phase cytocide removed these differences. Since a significant fraction of plateau-phase cells are not mitotically quiescent (Q), one must eliminate the proliferating (P) fraction if one wishes to examine the PLDR of the Q cells. For V79 cells, removal of the P cells eliminates the higher survival (usually interpreted as Q cell PLDR) of plateau-phase cells.

The addition of a lytic layer to a preformed linear sucrose gradient induces a temporary (up to half a day) initial gradient of considerable steepness which retards the sedimentation of large (>T4) DNAs. Centrifugation at sufficiently slow angular velocities permits the temporary initial gradient to disappear and therefore the sedimentation distance increases, yielding a rotor speed dependent effect on sedimentation distance. Gradients which are free of this effect are described and shown to permit mouse leukemia cell DNA to sediment independently of rotor speed (5–30 krpm).

Chinese hamster V-79 cells were exposed in suspensions for 1–15 min to 1.1 MHz continuous wave (CW) ultrasound at axial intensifies from 0.25–30 W/cm2. Cell lysis was evidenced by a decrease in the number of intact cells, and loss of reproductive integrity was evidenced by a decrease in the plating efficiency of the remaining intact cells. The magnitude of these effects was a function of both intensity and exposure duration. Mitotically synchronized cells displayed a differential sensitivity depending upon cell cycle position. The M and S phases of the cell cycle were more resistant with respect to cell lysis and loss of reproductive integrity than were the G1 and G2 phases.

The effect of solvent viscosity (ηs) and temperature (T) on the shape of the concentration dependence of the principal and total recoils in creep-recovery viscoelastometry experiments has been studied for T4 DNA solutions. The range of DNA concentration (c) was 2 – 40 μg/ml; glycerol, 70–80% v/v, sucrose, 60% v/v; NaCl, 5 mM – 1M; and T, 275 – 323 K. A linear proportionality between recoil and c was obtained at high ηs/T. At low ηs/T, the c-dependence was nonlinear, approaching saturation at higher c. At low c, the slope of both curves was the same. Transition between “linear” and “nonlinear” values occurred over a narrow range of ηs/T (a width of 1–5 K if ηs/T was changed by varying T). (ηs/T)tr, the midpoint of the transition, was independent of solvent properties other than viscosity. Also, (ηs/T)tr increased with c. For a given c, ηs/T values above this transitional value yield linear behavior; below this, nonlinear behavior. The ratio of linear to nonlinear recoil values is a linear function of c with Kc, the slope of this dependence, independent of ηs and T. A kinetic model for the observed nonlinearity of recoil with c is presented. It explains the independence of Kc on ηs and T. An attempt has been made to explain the linear–nonlinear transitions by comparison of τ1 and TR, the lifetime of the contact points of the polymer network in the de Gennes theory. The nonlinear values are consistent with a pseudogel that exists when τ1 TR, the DNA behavior is similar to that in dilute solutions (linear values). Thus, the condition for transition is τ1 = TR. However, some unsolved problems remain.

A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients.

The nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25 degrees C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37 degrees C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37 degrees C followed by 4 h holding at 25 degrees C prior to the second half of a split dose, or 5 h holding at 25 degrees C without a 37 degrees C incubation during the interval between doses. Longer incubations at 37 degrees C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37 degrees C was observed only during the first 30 min; thereafter, 3H-dThdR incorporation was higher than in unirradiated controls. The excess synthesis effect was removed by shifting irradiated cells to 25 degrees C holding. The inhibition observed at 25 degrees C was reversed by shifting to 37 degrees C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37 degrees C, and by 3 h at 25 degrees C; DNA double-strand break (DSB) repair plateaus at 80% (37 degrees C) and 60% (25 degrees C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.

The necessary conditions for a unique solution of the sedimentation vs DNA molecular weight equations are considered and applied to the native DNA of the L5178Y mouse leukemia cell. A brief review and critique of the literature of sedimentation anomalies is given to demonstrate that such anomalies are not present in the data reported here. It is shown that the chromosomal DNA of L5178Y cells comes in uniform packages of 1.0 (0.5–2.0) × 1010 daltons. All pieces are of an identical size which corresponds to the DNA content of about 1/13 the average chromatid. Both the size estimate and the number of such molecules/cell are confirmed by viscoelastometry. This DNA is shown to be free of radioactively demonstrable protein and/or lipid contaminants and of the same isopycnic density as T4 DNA. Variance analysis is applied to determine the precision of all measurements and to pinpoint major sources of error. A relationship between [η] and M is derived for native DNA in 1.0M NaCl. A necessary conclusion from these data is that mammalian chromosome models requiring degrees of polynemy greater than 16-neme (in G1) are incorrect (to the extent that the L5178Y cell is typical of mammalian cells).

Viscoelastometry enables one to determine both size (Mr) and number concentration (L1) of intact genome molecules in solution. Comparison of four parameters, corrected for shear stress, as functions of 60Co gamma-ray dose showed that (1) the principal retardation time (tau 11,0) remained constant, indicating that intact genomes (bacteriophage T4c) were being measured; (2) the principal recoil (gamma 11,r,0) decreased with dose directly proportionately to (and determining) L1; (3) both the total recoil (gamma r,0) and the recoil area (Ar,0), under conditions of high solvent viscosity decreased with dose almost as sensitively as gamma 11,r,0. The DNAD37 was 540 +/- 25 Gy and the biological PFUD37 was 410.1 +/- 4.5 Gy yielding 75.9 +/- 3.6% of inactivating events explicable by one double-strand break (DSB) per genome. This value is comparable to Freifelder's [Virology 36, 613-619 (1981)] value of 86% for phage T4r48+, and the Frankenberg-Schwager et al. (Br. J. Cancer, in press) value of 0.84 DSB/cell/lethal event in diploid mutant rad54-3 yeast under conditions restrictive for DSB repair. Therefore, T4c may be an excellent model system for DNA damage repair studies with relevance to pro- and eukaryotes. Viscoelastometry, working near its lower size limit, provided precise estimates of the proportion of genomes lacking DSBs. It is not subject to the molecular deformation upper size limitations of the other biophysically understood size measurement methods (e.g., sedimentation rotor speed dependence). Therefore, it should be the method of choice for the study of genomes larger than those of the T even bacteriophages.

The model presented here describes the DNA viscosity and viscoelastometric responses to ionizing radiation-induced molecular breakdown. Four DNA conformations are considered: (1) linear chain, (2) relaxed circle, (3) supercoiled circle, and (4) supercoiled linear. The model includes the systems of differential equations for calculating the size distributions of DNA molecules as functions of dose. Thedistributions are used in the usual hydrodynamic formulae for heterogeneouspopulations of polymer molecules. The sensitivity of viscoelastometry for the estimation of D37 is better than that of viscometry. The model predicts a considerable difference between the radiation responsesof linear and circular DNAs. The prediction is confirmed by experimental data for T4 DNA [C. S. Lange et al. (1984) Rad. Res.100, 1–15] and E. coli DNA [S. E. Bresler et al. (1984) Biophys. J.45, 749–754]. Thus, ionizing radiation can be used to determine the original conformation of DNA molecules.

SummaryAnalysis of the clone sizes of HeLa cells plated out by the Puck technique showed that the distribution broadened when the cell population included damaged cells with a slower net growth rate. This broadening of the clone-size distribution was found after repeated trypsinization of control cells as well as following single radiation doses and continuous irradiation. The development of these broad clone-size distributions has been studied over a number of days. The resultant families of distribution curves reveal additional data about the population kinetics of irradiated cells, which can be related to the growth curves and survival fractions obtained under the same experimental conditions.The more slowly growing clones, which develop after irradiation, have been shown to maintain proliferative capacity. The implications of this non-lethal radiation damage are discussed. It is emphasized that the usual parameter of a certain minimum clone size (usually 50 cells) is an arbitrary definition of the rep...

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 microM CPT enhanced the radiosensitivity of LY-S cells (D0 decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly (ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D0 decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e.g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases alpha and delta) applied concomitantly with CPT in cells not pretreated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication.

The planarian owes its extensive powers of regeneration to the possession of a totipotential stem cell system. The survival of the animal after irradiation depends mainly upon this system. In this respect the planarian is analogous to mammalian organ systems such as bone marrow or gut epithelium. The differentiated cells control the course of stem cell mediated tissue renewal by the secretion of differentiator and/or inhibitor substances. One such inhibitor substance, present in extracts prepared from homogenized whole planarians, specifically inhibits brain formation. This substance is organ specific, but not species specific. The differentiative integrity of the stem cells after irradiation is measured by comparing the regenerated brain volumes resulting from the presence or absence of the brain inhibitory extract during the regeneration period. Our data suggest that increasing doses of x irradiation decreases the ability of the stem cells to respond to differentiative substances. The data presented also explore the possibility of altering the postirradiation recovery pattern by shifting the differentiative demands placed on the stem cells. The final proportions of animals (one-half regenerated with, and one-half without, the extract) surviving after 60 days were not significantly different.

DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.

Potentially lethal damage (PLD) and sublethal damage (SLD) modification in L5178Y-S (LY-S) and L5178Y-R (LY-R) cells was investigated for postirradiation holding in either plateau phase or log phas...

Exposure of Chinese hamster V-79 cells to 1.1-MHz continuous wave (CW) ultrasound at intensities of 10, 20, and $30\ {\rm W}/{\rm cm}^{2}$ resulted in cell lysis and the loss of reproductive integrity (i.e., a decrease in plating efficiency) in the remaining intact cells. Sonication in the presence of 8 mM cysteamine, a free-radical scavenger, did not alter the amount of cell lysis, but did result in a smaller decrease in plating efficiency at 20 and $30\ {\rm W}/{\rm cm}^{2}$ . Hence, while free radicals do not appear responsible for ultrasonically induced cell lysis, free radicals do appear to be at least partially responsible for loss of reproductive integrity in the remaining intact cells.