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Previous studies have indicated that the nature of the interaction of radiation and Taxol may be dependent on the cell line, drug dose and treatment schedule. The present study represents a systematic attempt to examine the schedule dependence of the interaction of radiation and Taxol in HeLa cells. The protocol used was radiation treatment (7 Gy), followed by a variable interval (0-24 h), followed by exposure to Taxol (7.5 nM, 24 h), followed by plating for a colony-forming assay. Parallel samples were also taken for flow cytometric analysis of the distribution of cells in the phases of the cell cycle at the beginning and end of Taxol treatment. The results indicate that sub-additive, additive or supra-additive interaction can be seen depending on the interval between the radiation and Taxol treatments. Full radiation survival curves were determined for cells exposed to Taxol either immediately or 10 h after the completion of radiation treatment, corresponding to sub-additive and supra-additive interactions, respectively. An examination of the data revealed that maximum cell killing occurred when the percentage of cells in G1 phase was at a minimum at the time of addition of Taxol. Studies of Taxol-induced toxicity using cells synchronized in G1 phase with mimosine and then released and allowed to progress through the cell cycle confirmed this observation. The conclusion of this study is that prior radiation treatment can modify the effect of subsequent Taxol treatment through alterations in the distribution of cells in the phases of the cell cycle. This finding has important implications for the clinical scheduling of these two cancer treatment modalities.

The effect of reduced temperature (22 degrees C) or serum deprivation during low-dose-rate (0.66 cGy/min) gamma irradiation on cell killing and neoplastic transformation has been examined using the HeLa x skin fibroblast human hybrid cell system. The reduced temperature stops progression of these cells through the cell cycle while serum deprivation slows down cell turnover markedly. The data demonstrate an enhancement in both of the end points when cells are held at 22 degrees C compared to parallel experiments done at 37 degrees C. In operational terms, the decreased survival and increased neoplastic transformation are consistent with our earlier hypothesis of a higher probability of misrepair at reduced temperature (Redpath et al., Radiat. Res. 137, 323-329, 1994). The interpretation that this damage enhancement was associated with the reduced temperature, and not the fact that the cells were noncycling, was supported by the results of experiments performed with cells cultured at 37 degrees C in serum-free medium for 35 h prior to and then during the 12.24 h low-dose-rate radiation exposure. Under these conditions, cell cycle progression, as shown by reduction in growth rate and dual-parameter flow cytometric analysis, was considerably inhibited (cell cycle time increased from 20 h to 40 h), and there was no significant enhancement of cell killing or neoplastic transformation.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

A two- to threefold increase in the rate of neoplastic transformation in cells irradiated at a dose rate of 0.22 cGy/min with fission-spectrum neutrons compared to that at 10.7 cGy/min has been confirmed with the use of alkaline phosphatase chromogenic substrate Western Blue staining to detect foci of neoplastically transformed cells through their expression of a tumor-associated antigen, the end point of the HeLa × skin fibroblast human hybrid cell transformation assay. To investigate whether the inverse dose-rate effect is due to the existence of a period in the cell cycle in which cells are significantly more sensitive to neoplastic transformation than in the rest of the cell cycle, as has been postulated previously (Rossi and Kellerer, Int. J. Radiat. Biol. 50, 353-361, 1986; Brenner and Hall, Int. J. Radiat. Biol. 58, 745-758, 1990; Elkind, Int. J. Radiat. Biol. 59, 1467-1475, 1991), we compared the sensitivity of late mitotic/early G1-phase and $\text{mid-}{\rm G}_{1}\text{-phase}$

The HeLa x skin fibroblast human hybrid cell system has proven to be an excellent model system for quantitative studies of radiation-induced neoplastic transformation in vitro. A unique aspect of this system is the reexpression of a cell surface protein p75/150 with tumorigenicity. The identification of p75/150 as intestinal alkaline phosphatase (IAP) allowed for the recent development of a more simplified, rapid, and sensitive screening method than the previous p75/150 antibody-based staining procedure. The new method directly detects neoplastically transformed, IAP-expressing cells by staining with the alkaline phosphatase chromogenic substrate, Western Blue (WB). Earlier studies with the antibody-based immunoperoxidase assay indicated that, while no foci with tumor-associated antigen (p75-positive) were evident 15 days after irradiation, the number of foci rose quickly and leveled off between Day 19 and Day 23. This late appearance of the IAP-positive foci suggested that the neoplastic transformation process was not an immediate consequence of radiation damage. The mechanism underlying this observation was unknown. The possibility existed that very small foci and/or foci expressing a low level of IAP were being missed at earlier expression times. The increased sensitivity of the WB staining technique has allowed for the reinvestigation of the kinetics of induction of radiation-induced foci in this system. Experiments were performed where parallel groups of transformation flasks were stained at Days 7, 9, 11, 13, 15, 17, 19, and 21 days after irradiation. The data clearly indicate that the radiation induction of IAP-positive foci is indeed delayed in this system with the vast majority of the foci beginning to appear after Day 9 after irradiation. The delay is not the result of a lack of ability to detect small IAP-positive foci since foci with as few as 15 IAP-positive cells were discernible. We have reported previously that under identical experimental conditions both the establishment of plateau phase and the onset of the expression of lethal mutations also occur after Day 9. We therefore propose that radiation-induced neoplastic transformation of HeLa x skin fibroblast hybrid cells is a consequence of the delayed expression of heritable damage under epigenetic control with a resultant loss of tumor-suppressor function.

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

The HeLa × skin fibroblast human cell hybrid system has been used to study the effect of fission-neutron dose rate on the induction of neoplastic transformation. Previously published data using neu...

The dependence of γ-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa × skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 o...

The plating efficiency (PE) of a γ-irradiated (7 Gy) human cell hybrid line (HeLa × skin fibroblast, designated as CGL1) has been measured as a function of time postirradiation and compared to that of unirradiated cells at similar cell densities and under the same growth conditions. The results indicate that following irradiation, the PE of the irradiated cells initially increases but never returns to that of unirradiated cells during the experimental period that we have examined. Furthermore, after a period of 9 to 10 days (equivalent to at least 10 cell doublings) postirradiation and plating, the PE of the irradiated cells begins to decrease and continues to do so over the next 5 days. A decrease does not occur in unirradiated cells until much later (i.e., Day 15) corresponding to at least 5 additional cell doublings. The data are discussed in terms of a delayed expression of lethal mutations. The possible impact of these observations on the estimation of radiation-induced transformation frequencies is ...

The reduction of the disulfide bridge in the lipoate anion by peptide radicals and the formate radical ion (CO$sub 2$$sup -$) has been studied using the pulse radiolysis method. Peptide radicals were produced by the reaction of the hydrated electrons with the peptide. It was found that e/sub aq/$sup -$ reacts with diglycine, chloro-acetyl glycine and chloroacetyl diglycine to form a deaminated peptide radical (.CH$sub 2$CONHCH$sub 2$COR). The reaction of e/sub aq/$sup -$ with triglycine at neutral pH forms two radicals: (a) the deaminated radical which is formed when the e/sub aq/$sup -$ reacts with the carbonyl group in an $alpha$ position to the amino group, and (b) an ''electron adduct'' on the carbonyl group of the peptide bond which is formed when the e/sub aq/$sup -$ peptide reaction occurs at other positions. The ''electron adduct'' on the carbonyl radical can be formed by the reaction of the e/sub aq/$sup -$ with acetyl amide. The rate constant for reduction of the lipoate ion disulfide bridge by the deaminated radical was evaluated to be 5 x 10$sup 6$ M$sup - 1$sec$sup -$1, while that for ''electron adduct'' radical and the formate radical is approximately 5 x 10$sup 8$ M$sup -1$ sec$sup -1$. In view of these results a multistep mechanism is suggested for the e/sub aq/$sup -$ reduction of S-S bridge proteins. CO$sub 2$$sup -$ reacts directly with the disulfide bridge. (auth)

The radiation inactivation of T7 phage by 25-MeV electron pulses has been measured in various media containing a wide concentration range of radical scavenging solutes and in the presence of protective and sensitizing agents. The dependence of sensitivity on pulse dose, from 1 mrad to 3.6 krad, is attributed to radical depletion via bimolecular processes. The survival data are analyzed by extending target theory to include diffusive reactions of primary and secondary radicals generated in the medium. It is concluded that OH radicals are the principal primary inactivating species and that secondary radicals from Br/sup -/, CNS/sup -/, uracil, glucose, ribose, sucrose, tyrosine, and histidine are lethal to some extent. In nutrient broth or 100 mM histidine, psoralen derivatives, Actinomycin D, and Mitomycin C are anoxic sensitizers. It is proposed that the psoralens promote the formation of non-strand break lesions as the sensitization mechanism. The target theory based on diffusional kinetics is applicable to other systems including single cells.

Human skin fibroblasts are extremely refractory to neoplastic transformation by ionizing radiation [C. Borek, Nature 283, 776-778 (1980); M. Namba, H. Nishitani, and T. Kimoto, J. Exp. Med. 48, 303...

The neoplastic transformation of human cell hybrids (HeLa x skin fibroblasts) is accompanied by the expression of a cell surface protein for which monoclonal antibodies have been raised. The gamma-radiation-induced neoplastic transformation of these cells has been studied where the expression of this cell surface protein, as detected by immunoperoxidase staining, has been used as an end point. The yield of foci of positively staining cells has been shown to increase with increasing time postirradiation at which the assay is done and decrease with increasing density of viable cells plated postirradiation. The time of plating postirradiation is also an important parameter with transformation frequencies increasing over the first 6 h of postirradiation holding at confluence, followed by a gradual decrease.

The radiosensitivity of the E. coli fatty acid auxotroph K-1060 has been shown to increase with the number of carbon-carbon double bonds in the fatty acid used in the growth medium. This increase applies to bacteria irradiated under both anoxic and oxic conditions. It is suggested that the primary mechanism underlying these observations may involve a slow postirradiation peroxidative process.

The RBE of the Fermilab fast neutron beam (anti E = 25 MeV) relative to 6-MeV photons has been studied for gastrointestinal and hemopoietic syndromes in the mouse. Dose fractionation experiments have allowed characterization of the RBE as a function of neutron dose, and the calculation of cellular survival kinetic parameters and RBE coefficients. The data obtained indicate the Fermilab beam to have a biological effect which is very similar to that of the 50-MeV d ..-->.. Be TAMVEC beam.

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs γ irradiation. Furthermore, the data strongly suggest a more than additive interaction between the γ-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/107 survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by γ irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.

We have recently described a quantitative assay for the radiation-induced neoplastic transformation of human cell hybrids which is based upon the expression of a tumor-associated antigen (1, 2). In our studies of the kinetics of expression of this antigen, as assessed by the number of colonies or foci of cells detected using immunoperoxidase staining, no expression was seen for the first 15 to 17 days postirradiation (2). There then followed a constant level of expression up to 26 days postirradiation which was followed by a rapid continuous increase over the next few weeks (2). We suggested that this increase at times beyond 26 days could most likely be due to the formation of satellite colonies (2). This is not an unlikely possibility since these cultures are fed twice per week from Day 10 on. We have since carried out construction experiments to test this hypothesis. For the experiments reported here, the growth media and immunoperoxidase staining technique were as previously described (1, 2). The tumorigenic and the nontumorigenic cells used are designated as CGL3 and CGL 1, respectively. The derivation of these cell lines has previously been described (3). In these experiments, we mixed a known number (approximately two per T-25 flask) of tumorigenic cells (i.e., cells which express the tumor-associated antigen) with known numbers (84, 840, 8400, and 84,000 per T-25 flask) of nontumorigenic cells. The flasks were then stained using the immunoperoxidase technique at various times postmixing (7, 11, 21, and 28 days). The results are shown in Table I. It can be seen that at 7 days postmixing, the average number of positive colonies per T-25 flask was essentially equal to the estimated number of cells seeded. However, at 11 days postmixing, during which there were two refeedings (Days 5 and 8), there are on average a greater number of positively staining colonies than were seen at Day 7 a result which is consistent with satellite formation. This effect is much more noticeable at Day 21 (refeedings on Days 5, 8, 12, 15, 19) and Day 28 (refeedings on Days 5, 8, 12, 15, 19, 22, 26). Using a two-way analysis of variance on an individual flask basis, it was shown

ADAMS, G. E., BISBY, R. H., CUNDALL, R. B., REDPATH, J. L., AND WILLSON, R. L. Selective Free Radical Reactions with Proteins and Enzymes: The Inactivation of Ribonuclease. Radiat. Res. 49, 290-299 (1972). The effect of free radicals derived from thiocyanate, bromide, and carbonate ions on the activity of ribonuclease have been studied in neutral and alkaline solutions. Thiocyanate ions and, to a lesser extent, carbonate ions, protect ribonuclease against radiation-induced inactivation, although the presence of bromide ions has little effect. The absolute rate constants for the reactions of these radical anions with ribonuclease have been measured as a function of pH and salt concentration, and the transient absorption spectra of the reaction products have been determined. The results suggest that damage to a histidine residue, or residues, leads directly to loss of activity and support previous conclusions that exposed tyrosine residues in the enzyme are not essential for activity.

The radioprotection of dilute aqueous solutions of the enzyme papain, and of suspensions of Serratia marcescens by the sulfhydryl compound, dithiothreitol, has been demonstrated. The results are considered in terms of the stability of this compound to air oxidation and its free radical reactivity. It has been shown that radical scavenging and radical repair processes are the major causes of the observed radioprotective effect. The effect of medium and of the saturating gas on the radioprotective action of dithiothreitol toward Serratia marcescens is discussed.

ADAMS, G. E., BAVERSTOCK, K. F., CUNDALL, R. B., AND REDPATH, J. L. Radiation Effects on a-Chymotrypsin in Aqueous Solution: Pulse Radiolysis and Inactivation Studies, Radiat. Res. 54, 375-387, (1973). Pulse radiolysis and steady-state inactivation methods have been used to study the separate roles of H, OH, and e.q- in the radiation-induced inactivation of aqueous a-chymotrypsin solutions. The effects of nitrous oxide and t-butanol used as selective free radical scavengers allow the inactivating efficiencies per unit G-value of OH, H, and eaq- radicals to be estimated; these are 0.137, 0.13, and 0.055, respectively, in neutral solution. The transient spectra observed show that both OH radicals and hydrogen atoms react mainly with tryptophan residues. Only about 15% of the hydrated electrons react with the cystine disulfide linkages; rupture of some of these linkages may lead to inactivation. It is suggested that the protective action of oxygen is due partly to the reactions of 02 with enzyme radicals formed initially by reaction with OH.