Cultural adaptation of cognitive stimulation therapy (CST) for Chinese people with dementia: multicentre pilot study.

OBJECTIVE: Ageing of the Chinese population will drive a continued surge in dementia prevalence. Empirically tested non-pharmacological interventions developed in western cultures may be implemented in Chinese. Cognitive Stimulation Therapy (CST) that originated in the UK has proven benefits on cognition and quality of life in people with dementia. We investigated the feasibility and cultural appropriateness of CST in Hong Kong Chinese (CST-HK). METHODS: Mixed methods research was conducted following the formative method for adapting psychotherapy. A culturally adapted CST-HK, developed involving multidisciplinary stakeholders, was tested in a pilot multicentre study in people with mild dementia (n = 30) receiving community or residential care. Changes in cognition and quality of life were measured. Opinions from family caregivers and group facilitators (n = 25) were collected through focus groups and in-depth interviews for understanding the appropriateness of CST-HK. Feasibility was explored. RESULTS: After receiving CST-HK, 54% of participants achieved outcome of no cognitive deterioration, and 23% showed clinically meaningful improvement. Family caregivers and group facilitators expressed good acceptance of CST, with a low attrition (13%) and high attendance rate of CST-HK sessions (92%). Key cultural issues identified are (i) less active opinion sharing in group discussions due to conservatism/cautiousness and (ii) preference of practical activities with reward/recognition over pure discussion due to pragmatism. CONCLUSIONS: The CST-HK is feasible and culturally appropriate in Hong Kong Chinese. Further amendments can be made to ensure language use and enjoyment, with potential implications on effectiveness. We have provided a systematically developed, culturally adapted protocol for larger-scale implementation and research in Chinese populations. Copyright © 2017 John Wiley & Sons, Ltd.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability.

Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Detection of hypoxic cells in a murine tumor with the use of the comet assay.

BACKGROUND: Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents. PURPOSE: To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells. METHODS: Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radio-sensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells. RESULTS: After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method. CONCLUSIONS: Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors. IMPLICATIONS: Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.

Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells.

The comet assay, which measures DNA strand breakage in individual cells, was used to examine the relation between DNA damage, cell survival, and resistance to the topoisomerase II inhibitor etoposide (VP-16). Chinese hamster V79-171b cells and a VP-16-resistant subline (VPr) were exposed to VP-16 as monolayers or spheroids. The comet assay was comparable in sensitivity to the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks induced by VP-16. However, unlike conventional DNA damage assays, the comet assay also indicated heterogeneity in cell response. For V79 multicell spheroids exposed to VP-16, the external cycling cells were 50 times more sensitive to killing and DNA damage than the internal noncycling cells; the comet assay indicated the fraction of cells resistant to the drug. VPr cells, which were 10 times more resistant to killing and DNA damage by VP-16 than the parent cell line, could also be identified in mixed populations with the use of this method. These results suggest that the comet assay could be useful in predicting tumor cell response to DNA-damaging agents.

Correlation between metabolic reduction rates and electron affinity of nitroheterocycles.

Nitroheterocyclic compounds can selectively sensitize hypoxic (tumor) cells to radiation damage in vitro. However, results in vivo have generally been less optimistic, inasmuch as metabolic reduction of these drugs not only limits effective lifetime but also produces metabolic intermediates with marked cytotoxic and carcinogenic activity. With three reducing systems in vitro, E. coli B/r, mouse L-929 cells, and mouse liver microsomes, the rate of nitroreduction of several nitroheterocycles was found to be proportional to their electron affinity (half-wave reduction potential). Relative to the rate of nitrofurazone reduction in each system, metronidazole (Flagyl), N-hydroxyethyl-3,5-dinitropyrrole, misonidazole, nifuroxime, nitrofurantoin, and furylfuramide were metabolized about 200, 20, 2, 1.4, and 1.2 times less rapidly, while 3,5-dinitrobenzonitrile, 2,5-dinitrophenol, and 5-nitro-2-furaldehyde diacetate were reduced 2, 3, and 4 times more rapidly. Since nitroreduction has previously been correlated with subsequent cytotoxicity, DNA damage, and mutagenicity, the present results suggest that improvements in the therapeutic efficacy of nitroheterocycles (i.e., sensitization without toxicity and carcinogenicity) will be dependent on development of drugs with more appropriate pharmacological properties.

Multicell spheroid response to drugs predicted with the comet assay.

Multicell spheroids were exposed to DNA-damaging agents with the aim of determining whether prompt DNA damage could be predictive for cell killing and drug resistance. Chinese hamster V79 cells, SiHa human cervical carcinoma cells, and WiDr human colon carcinoma cells were grown as spheroids and exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), doxorubicin, etoposide, actinomycin D, 1-(2-nitro-1-imidazolyl)-3-aziridino-2-propanol (RSU 1069), 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine), and nitrogen mustard. Average DNA damage measured using the alkali comet assay generally correlated with cell killing irrespective of exposure times or drug concentration. However, better predictive power was achieved by using DNA damage levels in individual cells to identify the fraction of cells containing sufficient numbers of DNA strand breaks to cause death. Using this concept of a "threshold" for DNA damage, cell survival could be predicted for exposure to 4NQO, tirapazamine, nitrogen mustard, RSU 1069, and actinomycin D and was largely independent of cell type. The threshold value varied for each drug. For 4NQO, tirapazamine, and RSU 1069, DNA damage equivalent to about 10,000 strand breaks/cell was not toxic to cells of any spheroid type. Conversely, for actinomycin D, any DNA damage above background levels (approximately 100 breaks) was toxic for all three cell types. For some DNA-damaging drugs, the lack of correlation between DNA damage and cell killing was also informative. For etoposide and doxorubicin, no common threshold for cell killing could be determined, consistent with the hypothesis that DNA damage is only one of the actions of these drugs leading to cell death. For MNNG, the tail moment threshold varied significantly for the different spheroid types, probably indicating differences in repair. Overall, for five of the eight drugs, DNA damage measured using the comet assay was an effective and quantitative method of predicting drug cytotoxicity in complex multicelled systems.

Enhancement of toxicity from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea in V79 spheroids by a nitrofuran.

Hypoxic cell radiosensitizers enhance the cytotoxicity of several common chemotherapeutic agents in vitro and in vivo. Although this process has generally been called sensitization, few studies have documented true potentiation. We have used Chinese hamster V79 spheroids to study chemosensitization and fluorescence-activated cell sorting to specifically evaluate the roles of sensitizer binding and hypoxia in the effect. By using the median effect analysis to quantify the interactions of the agents, we conclude that marked potentiation can indeed be achieved. Somewhat greater potentiation was observed at increased depths within the spheroids, but the relative change was less than predicted on the basis of the decreasing oxygen tension. Further, increased toxicity did not necessarily lead to increased chemopotentiation, nor was potentiation directly related to the metabolism/binding of the nitrofuran. Thus, chemopotentiation is clearly a complicated process, highly dependent upon the sensitizer to antitumor drug ratio and the exposure conditions.

Reduction of tumor hypoxia and inhibition of DNA repair by nicotinamide after irradiation of SCCVII murine tumors and normal tissues.

The alkaline comet assay was used to measure both tumor hypoxic fraction and DNA strand break rejoining kinetics in individual cells from tumors and tissues of C3H/HeN mice exposed to ionizing radiation and nicotinamide. The percentage of hypoxic cells in SCCVII marine squamous cell carcinoma decreased from 18.4 to 4.4% in mice injected with a clinically relevant dose of 200 mg/kg nicotinamide 30 min before irradiation. At higher doses (500 and 800 mg/kg), nicotinamide also increased the half-time of strand break rejoining in tumor, thymus, spleen, bone marrow, and testis from 10-20 min to 40-80 min. Cells from the brain rejoined radiation-induced breaks 3-5 times more slowly than did cells from other tissues and showed no additional delay after nicotinamide. Cells with extensive numbers of strand breaks appeared 24 h after treatment with nicotinamide and radiation and 48 h after treatment with radiation alone. For most tissues, damage was more consistent with necrosis than with apoptosis. The percentage of heavily damaged cells was dependent on tissue type, time after irradiation, radiation dose, nicotinamide dose, and sequence of administration. In SCCVII tumors of air-breathing mice, nicotinamide enhanced radiation-induced cell killing primarily in cells close to the vasculature, but in tumors clamped before irradiation, 500 mg/kg nicotinamide did not increase cell kill. It appears that in addition to promoting tumor reoxygenation, nicotinamide inhibits DNA strand break rejoining in tumors and most normal tissues and promotes the earlier appearance of radiation-damaged cells, perhaps through inhibition of poly(ADP-ribose) polymerase.

Flow cytometry studies of intracellular adriamycin in single cells in vitro.

Flow cytometry techniques were used to quantify the intracellular fluorescence of Adriamycin in living cells. Additionally, fluorescence-activated cell sorting was utilized to investigate the relationship between intracellular drug concentration and cell viability. Uptake of Adriamycin in cultured cells was found to be highly dependent upon the method of cell growth (e.g., suspension versus monolayer cultures) and on cell density or cell crowding. With constant exposure conditions, however, direct proportionality was observed between mean cellular fluorescence and external Adriamycin concentration. Total cellular fluorescence was found to increase more rapidly than nuclear fluorescence, although both reached an apparent equilibrium in several hr. Under well-controlled exposure conditions, mean cell survival correlated well with mean cellular Adriamycin fluorescence. Similar results were observed for Adriamycin effects on DNA synthesis. Considerable heterogeneity of cellular Adriamycin levels was, however, observed in all cell populations, and fluorescence-activated cell sorting indicated that the cells with the least intracellular Adriamycin did indeed survive best.

Fluorescent nitroheterocycles for identifying hypoxic cells.

Binding of several nitroheterocycles by mammalian cells is a function of the ambient oxygen concentration; anoxic single cells bind up to 10 times as much of these drugs as do aerobic cells. We thus hypothesized that fluorescent nitroheterocyles could be used to quantitate the fraction of hypoxic cells in multicell systems, and this was tested in multicell spheroids using a relatively nontoxic nitrofuran, trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole. Binding of trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole, as quantified by flow cytometry, was highly responsive to external oxygen concentration. To assess the relevance of the observed fluorescence, fluorescence-activated cell sorting was used to examine the radiosensitivity of cells as a function of their fluorescence intensity. The most fluorescent were indeed the most radioresistant (i.e., contained the least oxygen). Additional results confirm the general feasibility of using fluorescent nitroheterocycles as hypoxic cell probes but also reveal that cellular binding of these agents is not exclusively dependent upon cellular oxygen content.

Damage to mammalian cell DNA by nitrofurans.

Maximum rates of nitrofuran reduction by intact mammalian cells and homogenates of mouse liver are obtained under anaerobic conditions, although significant reduction does occur with gas mixtures containing 5% oxygen and less. Single-strand breaks in DNA, measured as a decrease in the sedimentation constant on alkaline sucrose gradients, are produced in mammalian L929, KB, AND BHK-21 cells in vitro and Ehrlich ascites cells in vivo by several nitrofuran derivatives under hypoxic conditons.

Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links.

A new DNA precipitation assay used together with the alkali unwinding assay may provide a rapid means of detecting DNA damage in addition to strand breaks based on the relative amount of damage measured by the two assays. X-rays, Adriamycin, 4-nitroquinoline-N-oxide, N-methyl-N'-nitrosoguanidine, bleomycin, RSU 1172, and five other drugs produced the same relative amount of strand breakage by using the DNA precipitation and alkali unwinding assays. However, strand breaks produced by the bifunctional alkylating agents bis(2-chloroethyl)nitrosourea, RSU 1069, and RSU 1131 were detected with greater efficiency by the DNA precipitation assay, while the unwinding assay measured more strand breaks than the precipitation assay after damage by the topoisomerase inhibitors VP-16 and VM-26 and the DNA-condensing agents acridine orange and pyronin Y. Based on the reported mechanisms of action of these drugs, and studies with known DNA cross-linking agents, it appears that in addition to DNA strand breaks, the alkali unwinding assay is more sensitive to interstrand than to DNA-protein cross-links, while the DNA precipitation assay can be used to detect both types of cross-links. While quantification of specific lesions is not possible with this approach, the concomitant use of these two assays may provide a rapid and simple method for screening genotoxic drugs for DNA damage, and may also help to differentiate between DNA lesions which include strand breaks, interstrand and protein cross-links, DNA-phosphate adducts, and DNA-drug precipitates.

Intermittent blood flow in a murine tumor: radiobiological effects.

Little is known about how and why hypoxia arises in tumors, i.e., whether hypoxia is a chronic process resulting from diffusion limitations or occurs more acutely due to transient changes in blood perfusion. We have investigated the nature of hypoxia in the murine squamous carcinoma SCC VII using a new fluorescence-activated cell-sorting technique which facilitates isolation of viable tumor cells as a function of their distance from the blood supply. The technique utilizes the DNA binding/diffusion properties of the bisbenzamide fluorochrome Hoechst 33342. This compound has a very short distribution half-life from the blood after i.v. injection but remains bound within tumor cells even after disaggregation, redistributing with a half-life greater than 2 h. Cells can thus be sorted on the basis of their staining intensity (proximity to the blood supply), and varying the Hoechst 33342 administration protocol provides the basis for elucidating transient changes in blood flow that result in acute radiobiological hypoxia. Using this technique, we have demonstrated that acute hypoxia results from transient changes in blood perfusion in 500-mg SCC VII tumors. Independent confirmation of the intermittent blood flow has been obtained using histological techniques.

DNA double-strand breaks measured in individual cells subjected to gel electrophoresis.

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.

Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines.

The neutral filter elution method and the neutral comet assay have been used to analyze radiation-induced DNA damage and repair in 6 human tumor cell lines: HT-144 melanoma; DU-145 prostate carcinoma; U-87 glioma; WiDr and HT-29 colon adenocarcinomas; and SiHa cervical carcinoma. In spite of large differences in intrinsic radiosensitivity measured using a clonogenic assay, double-strand break induction, rejoining rate, and amount of residual DNA damage 4 h after irradiation were similar among these cell lines when measured using the neutral comet assay. Differences in initial numbers of double-strand breaks were observed using the neutral filter elution method; however, there was no correlation with radiosensitivity, nor did the rejoining rate or amount of residual DNA damage measured using filter elution correlate with radiation sensitivity. We conclude that neither double-strand break assay is able to reliably rank cells according to clonogenic survival following irradiation.

Use of the comet assay to identify cells sensitive to tirapazamine in multicell spheroids and tumors in mice.

Tirapazamine, a bioreductive drug preferentially toxic to hypoxic cells, produces significant numbers of DNA single-strand breaks that can be detected using the alkaline comet assay. Our goal was to determine whether single-strand breaks measured using this assay could act as a surrogate end point for cell killing in multicell spheroids and solid tumors in mice. In spheroids composed of Chinese hamster V79 cells, WiDr human colon carcinoma cells, or SiHa human cervical carcinoma cells, histograms of tail moments (indicators of DNA damage in the comet assay) could be used to identify the percentage of cells that sustained sufficient DNA damage to cause cell death after treatment with tirapazamine. The proportion of comets with tail moments