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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.

We have measured the clonogenic survival of cells isolated directly from colon cancer surgical specimens and treated with 5-fluorouracil (5-FU). Enzymatically dissssociated cells were incorporated into hybrid spheroids, consisting predominantly of nonproliferating HeLa feeder cells. Aliquots were exposed for 1.5 hr to a range of concentrations of 5-FU. From the decrease in clonogenic survival, as deduced from the frequency of colony formers among hybrid spheroids after chemical treatment, we were able to construct survival curves in 50% of the surgical specimens tested. A striking revelation was the presence of a resistant plateau in the survival curves, reminiscent of the solid tumor response to treatment with 5-FU. This resistance was absent in monolayer cultures. Evidence is presented that this resistance is due to the absence of, or delay in, cell cycle progression of cells residing in hybrid spheroids.

Abstract 2984 Background: Multiple myeloma (MM), a neoplasm of committed B-lymphocytes within the bone marrow (BM), is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in supporting tumor growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, we previously found EPCs from MM patients to be clonally restricted and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells (Braunstein et al., 2006). Based on the shared genetic identity that we and others have demonstrated between tumor cells and EPCs in MM patients, the present study explored the hypothesis that, similar to hemangioblasts, which are CD133-expressing precursors to adult hematopoietic and endothelial cells, EPCs may be a source of vascular and MM progenitor cells. Since hemangioblasts are known to exist predominately in the quiescent phases of the cell cycle, in this study we also examined the cell cycle status of CD133-expressing BM cells from MM patients in order to gain insight into their hemangioblastic traits. Methods: BM aspirates were acquired from MM patients under IRB approval. EPCs (>98% vWF/CD133/KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. The spleen colony assay was used to determine the stem cell capacity within BM-derived EPCs by i.v. injection into NOD/SCID mice. The spleens and BM of mice were harvested 2–4 weeks later. Cells were analyzed by immunofluorescence (IF) and flow cytometry. Hoechst 33342 (Hst) and Pyronin Y (PY) were used to measure the cell cycle status of CD133+ cells using FACS analysis. Results: Two to four weeks following i.v. injection of MM EPCs, human cell surface marker expression detected by flow cytometry within mouse BM and spleen cells shifted from CD133+/CD45-lo, a progenitor cell phenotype, to CD133−/CD45-hi, a more differentiated phenotype, suggesting the ability of MM EPCs to differentiate in vivo. Cell cycle analysis of the CD133+ population in BM cells of MM patients showed that these cells were predominantly non-cycling. On average, 60.5% of CD133+ cells were found to be in the G0/G1 phase of the cell cycle, as indicated by low levels of IF staining with Hst and PY. Conclusions: CD133+ cells are strong candidates as precursors to MM tumor and vascular progenitor cells. As quiescent cells are non-dividing, they often escape cytotoxic effects of chemotherapy, resulting in relapse, and therefore, identification of these cells is critical. Ongoing studies include the engraftment of CD133+ cells in the subcutaneous NOD/SCID gamma xenotransplant model and their growth in response to anti-myeloma strategies; results of these studies will be discussed. Disclosures: No relevant conflicts of interest to declare.

The Hybrid Spheroid assay is based on packaging tumor cells into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates provide an in vivo-like environment for entrapped test cells. 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 noncolony forming spheroids and the Poisson distribution, the average number of clonogens per spheroid can be calculated. The clonogenicity and radiation survival curves of cells derived from human tumors (of the maxilla, tongue, larynx, mouth floor, lung, breast, ovary, and colon) were so determined. Plating efficiency was increased in these normally poorly plating tumor cells, thus enabling survival measurements which are not practical using conventional methods. The Hybrid Spheroid assay has also been applied to determine the chemosensitivity of colon cancer cells.

Abstract 2816 Poster Board II-792 Background: Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM). Tumor growth and progression are promoted in MM through paracrine interactions between tumor cells, stromal cells, and matricellular components of the BM microenvironment. Endothelial progenitor cells (EPCs) are a key component of this microenvironment that mediate neovascularization. EPCs in MM patients contribute to neoangiogenesis and enhance the growth of tumor cells through the secretion of VEGF and IL-6. We found that EPC levels are increased in MM patients and covary with disease severity. Further, EPCs from MM patients were found by X chromosome inactivation studies to be clonally restricted, and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells. Based on new data regarding clonal restriction and on maintenance of MM EPCs in long-term culture compared both to EPCs obtained from normal controls and to human umbilical vein endothelial cells, the present study explored the hypothesis that EPCs in the vascular MM niche may be a source of MM-initiating cells with stem cell traits. The existence of MM stem cells is consistent with the finding of inevitable relapse of MM patients following response to treatment. We now show that clonal MM EPCs can be maintained in long term culture, unlike EPCs from healthy controls. In addition, our gene expression data show that MM EPCs have a higher level of expression of genes associated with stem cell function (e.g., GREM1, RUNX2, HOXC6, FOXF2) compared to the same patients' MM tumor cells, as well as to control EPCs. Genes such as WNT5A, SOX9, and ADAM12, which are involved in differentiation, an integral stem cell process, were also highly expressed in MM EPCs (manuscript in preparation). Stem cells have the abilities of self-renewal and differentiation. Cancer stem cells (CSCs), such as those found in acute myeloid leukemia, share these characteristics. Similar to hematopoietic stem cells (HSCs), CSCs are known to engraft to spleen and BM, where they proliferate and are capable of forming spleen colonies. To determine whether MM EPCs were able to home and proliferate in a similar fashion, we intravenously injected them into non-lethally irradiated mice. Using flow cytometry we examined the prevalence of human EPCs that expressed differentiation markers. The formation of spleen colonies was also assessed. Methods: EPCs (>98% vWF/CD133/ KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. Nine irradiated NOD/SCID mice were injected (i.v.) with 106 EPCs derived from 6 patients newly diagnosed with advanced MM. Spleens and BM were harvested 2-4 weeks later. The cells were analyzed by immunofluorescence and flow cytometry to determine the presence of human cell differentiation markers, including CD45 (a hematopoietic differentiation marker) and CXCR4 (a differentiation/migration marker). Results: EPCs cultured from 4 newly diagnosed patients were maintained in culture up to 7 months. Flow cytometry showed the engraftment of human cells to the spleen and BM of all mice 2-4 weeks following injection. The mean number of CD45+ cells in the experimental group increased within the spleen by 54.1 fold and in the BM by 35.9 fold compared to vehicle-injected controls. Similarly, CXCR4+ cells in the experimental group increased within the spleen by 15.9 fold and in the BM by 12.5 fold. In addition, of the 9 spleens harvested, 1 spleen showed colony formation, while none of the vehicle-treated animals displayed colony formation. Conclusion: Results show a high degree of proliferation of CD45+ cells within the spleen and BM following injection of MM EPCs. Since CD45 is a marker of hematopoietic differentiation, the proliferation of cells expressing this marker suggests that human hematopoietic cells expanded after EPCs were injected. Furthermore, the proliferation of cells expressing CXCR4+, which is required for HSC quiescence and which also plays a role in EPC differentiation, indicates that EPCs are part of the stem cell niche in MM. In addition to the sizeable increase seen in the number of cells expressing these differentiation markers, 1 of 9 mice injected, formed spleen colonies. Taken together, these data suggest that a portion of the MM EPC population possesses traits similar to those of HSCs and CSCs, and this thesis warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Purpose: To study the influence of combinations of Linear Accelerators and treatment planning‐systems, anatomical site and PTV volumes on MUs, and the magnitude and direction of deviation of prostate and H&N IMRT plan QA dose from plan‐dose. Method and Materials:IMRT plans were generated using Pinnacle3 or Eclipse treatment planning‐systems for treatment with Elekta or Varian Linacs, respectively. There were 29 H&N plans for Varian, and 59 and 33 prostate plans for Varian and Elekta, respectively. Dose at a point in the MED‐TEC phantom was measured with MT‐ERI‐A12 ion chamber. QA results were calculated as percentage deviation between measured and calculated doses in the phantom. A deviation of ±5% was acceptable. The magnitude and direction of deviation of QA results vs. PTV volumes, treatment site, Linac and planning system combinations were studied. PTV volumes vs. MUs was also analyzed. Linear regression analysis was used to study the relationship between paired variables. Results: The direction and magnitude of QA result deviation was Linac dependent. For Elekta prostate cases, QA measured dose deviated to negative direction (−2.49±0.93, −0.5 to −5%). For Varian cases, QA measured dose for prostate (1.67±0.91, −0.1 to 4.9%) and H&N cases (3.58±1.80, 0.2–6.4%) deviated to positive side. These results suggest that the delivered dose could vary between Linacs and might result in under‐ or over‐dosing. Prostate QA result deviation was independent of PTV volumes (P>0.1), but MUs increased with increase in PTV volumes (P

DNA repair capacity has been suggested to be a genetic mechanism which influences the rate of ageing and length of life. We recently reported an age-related decline in DNA double-strand break (DSB) repair in unstimulated human lymphocytes (Mayer, Lange, Bradley, and Nichols 1989, 1990). In that work peripheral lymphocytes isolated from whole blood donated by 20 normal, healthy subjects aged 23-78 years, were X-irradiated (30 Gy) on ice and incubated at 37 degrees C for repair times of 15, 30, and 120 min, and neutral filter elution was used to assay DSB induction and completeness of DSB rejoining. Further analysis reveals that the decrease in DSB rejoining appears to be more pronounced in older women than in older men (and may begin after age 65 years). Moreover the reported age-related decline in DSB induction occurs more rapidly (by a factor of ca. 2) in women than in men. We had previously demonstrated that, independently of in vivo age, cells with lower radiosensitivity (i.e. lesser DSB induction) appear to be less repair-proficient (Mayer et al. 1990). The present analysis reveals a gender-specific pattern in this correlation between extent of DSBs induced and percentage of DSBs rejoined: at comparable levels of DSB induction, cells from men rejoin a higher percentage of DSBs than do cells from women. This preliminary study suggests the following hypothesis: age-related DSB effects (i.e. induction and rejoining) are due to changes in chromatin structure and males are less sensitive than females to the influence which age-related alterations in chromatin exert on DSB effects.

Using the newly developed hybrid spheroid system, we have been able to measure sensitivities to adriamycin in two sublines of LoVo cells (a human colon adenocarcinoma line). Hybrid spheroids are composed of coagglomerated LoVo cells, and nonproliferating HeLa "feeder" cells, mimicking a population of minitumors. Spheroids of uniform size, but containing different numbers of LoVo cells, were incubated with various concentrations of adriamycin and after washing, plated for colony formation. Binomial counting statistics were achieved by marking surface-attached spheroids soon after plating, and scoring colonies one week later. From the fraction of noncolony formers, the average number of clonogens per spheroid (clonogenicity) was calculated using the zero term of the Poisson distribution. The effect of treatment with adriamycin was evaluated from the decrease in clonogenicity in parallel series of hybrid spheroids. Response to treatment was found to be independent of cellular clonogenicity in the control, untreated series. Dose-response curves were obtained for each of the two sublines after treatment for 1.5 h with various concentrations of adriamycin. The difference in the response to adriamycin between the two sublines amounted to a factor of 27 (deduced from D0 ratios; these are the same as isosurvival dose ratios).

If DNA repair plays a major role in aging (Gensler and Bernstein, 1981; Hart et al., 1979; Little, 1976; Yielding, 1974; recently reviewed in Warner et al., 1987), within a species one would expect to see decreased efficacy of repair processes in older animals. Some data support this prediction, whereas others do not (recently reviewed in Tice and Setlow, 1985). In general, as studied in nonhuman animals, in vivo age-related DNA repair capacities differ by type of damage, by types of repair, by species, and by strain as well as by organ or tissue (Licastro and Walford, 1985; Niedermuller et al., 1985; Su et al., 1984; Tice and Setlow, 1985; Vijg, 1987).

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.

We studied the influence of trypsin-induced morphological changes on the radiosensitivity of cells plated at either low (4-600/cm2) or high (2 x 10(4)/cm2) density and grown overnight before treatments. Trypsin treatment induced contraction and rounding of spread cells. The radiosensitivity of cells trypsinized and plated either: (1) immediately before [(a) D0 = 1.7 Gy for cells at low-, and (b) 1.5 Gy at high-density] or (2) immediately after (D0 = 1.6 Gy, high-density cells) irradiation was higher than that of (3) cells at high density, irradiated and delayed plated [cells remained spread until the completion of potentially lethal damage repair (PLDR) and trypsinization, D0 = 2.2 Gy], and (4) cells at low density which were neither delayed plated nor trypsinized (i.e. remained spread) after irradiation (Do = 2.4 Gy). These data show that PLDR is reduced in trypsin-treated cells of both high (compare 1b and 2 with 3) and low (compare 1a with 4) density cultures; the latter comparison provides a direct measure of the trypsin effect. Since the comparison between conditions 1 and 2 vs. 3 and 4 is of round vs. spread cells, PLDR appears to be influenced by the cell's morphological state. Kinetic studies showed that when cells were incubated in growth medium to recover from trypsin-induced effects before irradiation, the radiation sensitivity of spread cells (plated in situ), but not of those remaining rounded (in suspension until plating and irradiation), decreased and became equal to that of delayed plated high-density cells. Neither irradiated cells treated with hypertonic saline, nor mitotic cells, showed the trypsin effect. From these results we suggest that: (1) trypsin-induced cell contraction affects the ability of cells to repair radiation damage, (2) spread cells are better able to repair PLD than rounded cells, (3) immediate plating survival of cells in high-density cultures may not represent their intrinsic radiosensitivity and (4) cell-to-cell contact is not necessary for log phase cells to repair PLD.

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.

A substance which inhibits brain formation in decapitated regenerating planarians {Dugesia etrusca) was characterized and partially purified. The substance’s inhibitory activity was followed during each purification procedure by adding freshly decapitated animals of a standard size to each fraction, and later measuring the resultant regenerated brain volume. The inhibitory activity remained in the supernatant after a 10000 g centrifugation of a cell-free homogenate. Most of the activity sedimented when the 10000 g supernatant was centrifuged at 32000 g. The degree of inhibitory activity increased with increased numbers of animals in the initial homogenate. The substance has an apparent molecular weight between 2x 105 and 4x 105 daltons. Digestion by pronase destroyed the activity, but treatment with RNase, DNase I, or lipase had no significant effect. The inhibiting substance has an isoelectric point (pl) of between 4·75 and 5·38 and migrates to the anode when electrophorezed in pH 6·8 buffer.

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.

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.

Sedimentation coefficients of T7, T2H and T4 DNA were determined with isokinetic sucrose gradients in both 0.1 M and 1 M NaCl. The s values were completely equivalent to those measured by analytical ultracentrifuge and no reduction of s20,w was observed due to the presence of sucrose (anomalous sedimentation). s20,w values are calculated on the basis of both partial specific volume ( \ gn) and apparent specific volume (ϕ′). Using the latter value s20,w-molecular weight relations are derived for 0.1 M and 1 M NaCl solvents. The glucosylation of T2H and T4 DNA appears to influence s20,w in a manner disproportionate to the molecular weight added by glucose.

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.

SummaryA model is presented which defines the parameters of the curve for neoblast (stem cell) survival of reproductive integrity in terms of the parameters of the planarian radiation mortality curve. Two constants of the model, namely, the initial number of neoblasts (N) and the repopulation probability (α) for the independent repopulation of the irradiated planarian tissues by surviving neoblasts are defined and, for the species Dugesia lugubris, measured. The assumption that the repopulation probability for each neoblast is independent of the number of neoblasts is confirmed experimentally for diploid and triploid neoblasts of exogenous and endogenous origin. The repopulation probability for diploid (endogenous and exogenous) and triploid (endogenous only) neoblasts are not significantly different. Triploid exogenous neoblasts, however, are only 16 per cent as effective as endogenous triploid or exogenous diploid neoblasts. Values of D0 and NαE (E = extrapolation number) are given for diploid, triploid...

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.

Monolayer cultures of JU56 wallaby cells were exposed to germicidal U.V. and/or photoreactivating (PR) light. The U.V. exposures induced dose-dependent cell-death. The survival data are consistent with a common extrapolation number (n) of 6 x 17 +/- 0 x 98 with a D(0) of 123 x 0 +/- 6 x 8 erg/mm2 for photo-reactivated cells and a D0 of 87 x 3 +/- 4 x 9 erg/mm2 for non-photoreactivated cells; the photoreactivation protected the cells with a dose-modification factor of 1 x 41 +/- 0 x 02. Therefore PR is not a shoulder phenomenon and so has no relationship to the repair of sub-lethal damage.

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 literature on planarian ageing is briefly reviewed to distinguish those features which are symptoms of senility from those which are features of normal growth and development. The recommendation of Abeloos, to seek the causes of ageing in those processes of normal development which lead to cessation of growth, has been followed and the relationships between planarian chronological age and size, number of stem cells (neoblasts) and tissue density of stem cells have been examined. Quantitative data are presented on these parameters for the species Dugesia lugubris (O. Schmidt). The relevance of the drastic decrease in tissue neoblast density as the planarian increases in size is discussed in terms of the role of the neoblast in normal tissue renewal and in regeneration. It is concluded that although cumulative differentiation errors may contribute to the symptoms of senility, neoblast availability for regeneration and repair is probably the critical factor in planarian survival and that a shortage of neoblasts may even be the cause of the improper differentiation control by the differentiated tissues which results in the symptoms of senility.

SummaryA method is described for the estimation of a quantity which can be considered to be the potential ability to repair sub-lethal damage, or its equivalent, the amount of sub-lethal damage induced. This method was applied to populations of HeLa cells synchronized by mitotic harvest. The sensitivity (with respect to loss of reproductive integrity) of HeLa cells to single radiation doses and the ability to repair significant amounts of sub-lethal damage were found to be dependent on cell-cycle position. The more resistant phases were found to be associated with a greater potential for repair of sub-lethal damage. Significant repair was found to be possible only during early and mid G1 and in late S. The proportion of sub-lethal damage actually repaired was dependent on the fractionation interval. 4–8 h (⅙–1/3 of cell cycle) allowed only partial repair; 18 h (¾ of cell cycle) sufficed for full repair. 20–25°C incubation was found to permit progression from M into early G1, but to inhibit totally the rep...

The effect of chloramphenicol on progression through the cell cycle of L5178Y cells was investigated. Using eosin staining as a viability index, G2 cells were shown to be specifically killed at a concentration of chloramphenicol generally used to study mitochondrial protein synthesis. Pretreating cells with chloramphenicol induced resistance to this G2 lethality.

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.