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A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984 (Biochem. Biophys. Res. Commun. 123, 291–298). Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a “comet” with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate “features” of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor c...

The single-cell gel electrophoresis or "comet" assay was developed many years ago to analyze DNA damage in individual cells. It is a powerful and versatile technique that relies on microscopic visualization or imaging of DNA after single cells are embedded in agarose, lysed, and electrophoresed. In addition, the basic methodology has been extended to permit the detection of a variety of classes of DNA damage with good sensitivity in virtually any single-cell type. A unique but understudied property of the comet assay is its ability to detect and quantify cellular heterogeneity in response to DNA-damaging agents. This review outlines the considerations in producing and analyzing comet data when heterogeneity in induction of or cellular response to DNA damage is the major consideration. Examples are presented to emphasize the heterogeneity of tumor response to ionizing radiation and cytotoxic drugs.

We believe that names have a powerful influence on the experiments we do and the way in which we think. For this reason, and in the light of new evidence about the function and evolution of the vertebrate brain, an international consortium of neuroscientists has reconsidered the traditional, 100-year-old terminology that is used to describe the avian cerebrum. Our current understanding of the avian brain - in particular the neocortex-like cognitive functions of the avian pallium - requires a new terminology that better reflects these functions and the homologies between avian and mammalian brains.

Various studies have identified a critical role for Notch signaling in cardiovascular development. In this and other systems, Notch receptors and ligands are expressed in regions that undergo epithelial-to-mesenchymal transformation. However, there is no direct evidence that Notch activation can induce mesenchymal transdifferentiation. In this study we show that Notch activation in endothelial cells results in morphological, phenotypic, and functional changes consistent with mesenchymal transformation. These changes include downregulation of endothelial markers (vascular endothelial [VE]-cadherin, Tie1, Tie2, platelet-endothelial cell adhesion molecule-1, and endothelial NO synthase), upregulation of mesenchymal markers (α-smooth muscle actin, fibronectin, and platelet-derived growth factor receptors), and migration toward platelet-derived growth factor-BB. Notch-induced endothelial-to-mesenchymal transformation does not seem to require external regulation and is restricted to cells expressing activated Notch. Jagged1 stimulation of endothelial cells induces a similar mesenchymal transformation, and Jagged1, Notch1, and Notch4 are expressed in the ventricular outflow tract during stages of endocardial cushion formation. This is the first evidence that Jagged1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that Notch signaling may be required for proper endocardial cushion differentiation and/or vascular smooth muscle cell development.

The ability to imitate complex sounds is rare, and among birds has been found only in parrots, songbirds, and hummingbirds. Parrots exhibit the most advanced vocal mimicry among non-human animals. A few studies have noted differences in connectivity, brain position and shape in the vocal learning systems of parrots relative to songbirds and hummingbirds. However, only one parrot species, the budgerigar, has been examined and no differences in the presence of song system structures were found with other avian vocal learners. Motivated by questions of whether there are important differences in the vocal systems of parrots relative to other vocal learners, we used specialized constitutive gene expression, singing-driven gene expression, and neural connectivity tracing experiments to further characterize the song system of budgerigars and/or other parrots. We found that the parrot brain uniquely contains a song system within a song system. The parrot "core" song system is similar to the song systems of songbirds and hummingbirds, whereas the "shell" song system is unique to parrots. The core with only rudimentary shell regions were found in the New Zealand kea, representing one of the only living species at a basal divergence with all other parrots, implying that parrots evolved vocal learning systems at least 29 million years ago. Relative size differences in the core and shell regions occur among species, which we suggest could be related to species differences in vocal and cognitive abilities.

International audience; A new procedure of stratified sampling is proposed in order to establish an accurate estimation of Varroa destructor populations on sticky bottom boards of the hive. It is based on the spatial sampling theory that recommends using regular grid stratification in the case of spatially structured process. The distribution of varroa mites on sticky board being observed as spatially structured, we designed a sampling scheme based on a regular grid with circles centered on each grid element. This new procedure is then compared with a former method using partially random sampling. Relative error improvements are exposed on the basis of a large sample of simulated sticky boards (n = 20,000) which provides a complete range of spatial structures, from a random structure to a highly frame driven structure. The improvement of varroa mite number estimation is then measured by the percentage of counts with an error greater than a given level.

Tumor cells at very low oxygen tensions are known to be about three times more resistant to killing by ionizing radiation. Since cells at intermediate oxygen tensions (defined here as greater than 0.1% and less than 2% O(2)) show partial radioresistance, they should be a consideration in tumor treatment. In an effort to estimate the extent and range of oxygenation in SiHa human cervical carcinoma xenografts, patterns of cell killing and DNA damage by radiation and two bioreductive drugs, PD-144872 and RSU-1069, were compared to those seen in SiHa cells grown as spheroids. These drugs produce DNA interstrand crosslinks that are largely responsible for cell killing, and the degree of crosslinking increases as the oxygenation is reduced. About 60% of the cells in SiHa xenografts exhibited drug-induced crosslinks, but only about 35% showed extensive crosslinking indicative of hypoxia below 0.1% oxygen. Patterns of toxicity and DNA damage in xenografts were comparable to those of spheroids equilibrated with about 2% oxygen, indicating that most cells in the xenografts exhibit some radioresistance due to lack of oxygen. Similarly, pimonidazole binding indicated that about 60% of the cells in SiHa xenografts were either intermediate in oxygenation or hypoxic, but only about half of those were consistent with extreme oxygen depletion. The apparent size of the population of "intermediately hypoxic" cells has implications for the use of ionizing radiation, hypoxic cell cytotoxins, and other antitumor agents whose cytotoxicity is dependent on cellular oxygen content.

The perfusion of human tumour xenografts was manipulated by administration of diltiazem and pentoxifylline, and the extent that observed changes in tumour perfusion altered tumour radiosensitivity was determined. 2 tumour systems having intrinsically different types of hypoxia were studied. The responses of SiHa tumours, which have essentially no transient hypoxia, were compared to the responses of WiDr tumours, which contain chronically and transiently hypoxic cells. We found that relatively modest increases in net tumour perfusion increased tumour cell radiosensitivity in WiDr tumours to a greater extent than in SiHa tumours. Moreover, redistribution of blood flow within WiDr tumours was observed on a micro-regional level that was largely independent of changes in net tumour perfusion. Through fluorescence-activated cell sorting coupled with an in vivo–in vitro cloning assay, increases in the radiosensitivity of WiDr tumour cells at intermediate levels of oxygenation were observed, consistent with the expectation that a redistribution of tumour blood flow had increased oxygen delivery to transiently hypoxic tumour cells. Our data therefore suggest that drug-induced changes in tumour micro-perfusion can alter the radiosensitivity of transiently hypoxic tumour cells, and that increasing the radiosensitivity of tumour cells at intermediate levels of oxygenation is therapeutically relevant. © 2001 Cancer Research Campaign http://www.bjcancer.com

A combination of antitumor approaches acting on different death pathways seems ideal for increasing therapeutic responses, especially when defined resistance mechanisms interfere with individual cellular processes. Apoptosis pathways triggered by ionizing radiation (XRT) and the death ligand TRAIL were analysed in Jurkat lymphoma cells. Both induced the activation of caspase-8, caspase-3, BID and mitochondrial potential loss. TRAIL induced apoptosis required caspase-8, whereas it was not essential for radiation induced apoptosis. The inhibition of mitochondrial damage by Bcl-2 abrogated XRT induced apoptosis and caspase activation, but only marginally attenuated TRAIL induced cell death. The combined treatment with TRAIL and XRT exerted additive apoptotic effects in control cells, whereas highly synergistic effects occurred in cells overexpressing Bcl-2. In addition, a strong effect of TRAIL on radiation induced clonogenic cell death was found. In conclusion, TRAIL seems to be of high potential value for a combination with ionizing radiation in tumor therapy.

The distributions of calcitonin gene-related peptide (CGRP) immunoreactive neurons and fibers were mapped within forebrain vocal control and auditory nuclei of a vocal learning psittacine species, the budgerigar (Melopsittacus undulatus). Immunoreactivity was exhibited by telencephalic nuclei previously associated with vocal control pathways on the basis of both tract tracing studies and gene mapping: the central nucleus of the anterior archistriatum (AAc), central nucleus of the lateral neostriatum (NLc), magnocellular nucleus the lobus parolfactorius (LPOm), the oval nucleus of the ventral hyperstiratum (HVo) and the medial division of the oval nucleus of the anterior neostriatum (NAom). The main body of NAo also contained an exceptionally high density of immunoreactive fibers. In contrast to the condition in oscine songbirds, CGRP-positive neuronal somata were not present in any telencephalic vocal control nucleus. CGRP-positive somata were present, however, in diencephalic cell groups that included the shell region of the nucleus ovoidalis (Ov), the nucleus dorsolateralis posterior (DLP) and a region of the ventral thalamus that was retrogradely labeled by tracer deposits into HVo and AAc. CGRP immunoreactive fibers were observed within auditory areas of the telencephalon including Field L and the neostriatum intermedium pars dorsolateralis. The likely sources of these fibers are CGRP-positive neurons within the Ov shell and DLP.

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define ‘positive’ cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 ± 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m–2 pimonidazole also suggest the suitability of this approach for human tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com

The brain of the budgerigar (Melopsittacus undulatus), a small parrot that acquires new vocalizations throughout life, was examined for immunoreactivity to the opioid peptide methionine enkephalin (mENK). mENK is a highly prominent feature of the chemical architecture of the forebrain vocal system of oscine songbirds. Forebrain vocal control nuclei are believed to have evolved independently in parrots and songbirds (Streidter [1994] J. Comp. Neurol. 343:35-56); however, recent studies have found similarities in the neural organization of vocal control pathways in budgerigars and songbirds (Durand et al. [1997] J. Comp. Neurol. 377:179-206). Among the similarities are the existence of recursive pathways interconnecting vocal control neurons in the archistriatum, basal ganglia (i.e., lobus parolfactorius), and dorsal thalamus. In the present study, we found that all vocal control nuclei within the budgerigar forebrain exhibit prominent mENK-like immunoreactivity (ELI) in fibers and somata. We also found striking similarities between the morphology of ELI elements in budgerigar vocal control nuclei and that described previously in songbird vocal nuclei. Despite these similarities, the budgerigar dorsal striatopallidum (lobus parolfactorius, paleostriatum augmentatum, and paleostriatum primitivum) and somatomotor (anterior) archistriatum exhibit unique patterns of ELI. The dorsal striatopallidum contained far less ELI, whereas the archistriatum contained far more than would be expected on the basis of previous studies of opioid peptides in other avian species, including pigeons, chickens, and songbirds. These differences may reflect neural specializations unique to the budgerigar that contribute to the extraordinary flexibility of the vocal motor system of this species to acquire socially significant stimuli throughout life.

Background and purpose : The objective of the present study was to evaluate the predictive potential of cell kinetic parameters and repopulation rates determined by flow cytometry during multifraction irradiation of spheroids, a system in which the fate of all cells can be determined with high precision. Ultimately, similar analytical techniques should provide a reproducible and prognostically significant clinical predictive assay. Materials and methods : Multicellular spheroids of Chinese hamster V79 lung cells were irradiated with 2.5 Gy of 250 kVp X-rays twice daily to a total dose of 25 Gy. Repopulation parameters and cell kinetic parameters were followed throughout the irradiation period and for 5 days after completion of exposure. Results : (1) Regrowth (RG) took place early during multifraction irradiation. (2) Potential doubling time ( T pot ) decreased steadily from the early part of treatment, remaining of short duration until the spheroids almost attained the pre-treatment number of clonogenic cells. (3) Accelerated repopulation was mainly due to a decreased cell loss factor ( Φ ) and increased growth fraction (GF), although a modest decrease in cell cycle time ( t c ) was suggested. (4) Φ decreased during exponential RG. (5) Other parameters such as observed doubling time ( t d ) and labelling index (LI) paralleled these findings. Conclusions : Clonogen repopulation that began early in the irradiation scheme and accelerated rapidly is not consistent with the prevailing view that accelerated repopulation begins several weeks into clinical protocols. Also, pre-treatment T pot did not adequately estimate the repopulation speed in the spheroids. Equivalent studies in animal tumour systems, and then in the clinic, are consequently indicated and of some urgency.

Chinese hamster V79 monolayers, V79 spheroids, and SCCVII murine tumours were examined for DNA damage using the alkaline comet assay and for cell killing by measuring clonogenicity following a 1-h exposure to doxorubicin, N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG), 4-nitroquinoline- N -oxide (4-NQO), etoposide, or 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine). Greater heterogeneity in DNA damage was evident in spheroids compared to monolayers exposed to these drugs, and cell survival was correlated with the fraction of cells which lacked sufficient DNA damage following treatment with tirapazamine or doxorubicin. Cell sorting experiments verified that subpopulations of cells resistant to DNA damage were also more resistant to cell killing. Significant heterogeneity was observed in cells from SCCVII tumours exposed to tirapazamine and etoposide, and comet DNA content was used to independently assess DNA damage to aneuploid tumour cells and diploid host cells. These results suggest that, for some drugs, the comet assay may be an effective method of identifying drug-resistant cells in solid tumours.

A feature of the telencephalic vocal control system in the budgerigar (Melopsittacus undulatus) that has been hypothesized to represent a profound difference in organization from the oscine vocal system is its reported lack of an inherent circuit through the anterior forebrain. The present study reports anatomical connections that indicate the existence of an anterior forebrain circuit comparable in important ways to the “recursive” pathway of oscine songbirds. Results from anterograde and retrograde tracing experiments with biocytin and fluorescently labeled dextran amines indicate that the central nucleus of the anterior archistriatum (AAc) is the source of ascending projections upon the oval nuclei of the anterior neostriatum and ventral hyperstriatum (NAo and HVo, respectively). Efferent projections from the latter nuclei terminate in the lateral neostriatum afferent to AAc, thereby forming a short recurrent pathway through the pallium. Previously reported projections from HVo and NAo upon the magnocellular nucleus of the lobus parolfactorius (LPOm), and from LPOm onto the magnocellular nucleus of the dorsal thalamus (DMm; G.F. Striedter [1994] J. Comp. Neurol. 343:35–56), are confirmed. A specific projection from DMm onto NAom is also demonstrated; therefore, a recurrent pathway through the basal forebrain also exists in the budgerigar vocal system that is similar to the anterior forebrain circuit of oscine songbirds. Parallels between these circuits and mammalian basal ganglia-thalamo-cortical circuits are discussed. It is hypothesized that vocal control nuclei of the avian anterior neostriatum may perform a function similar to the primate supplemental motor area. J. Comp. Neurol. 377:179–206, 1997. © 1997 Wiley-Liss, Inc.

Tirapazamine, a new bioreductive agent currently advancing through clinical trials, may have a valuable role to play in cancer therapy. In vitro, the drug shows markedly more toxicity to hypoxic cells than to aerobic cells, and preferential activity against hypoxic cells of solid tumors in vivo also can be inferred in many investigations. However, we have previously reported that tirapazamine has minimal activity against cells in the center of hypoxic spheroids, raising concerns with regard to whether the drug may be bioreductively inactivated before reaching chronically hypoxic tumor cells. We consequently examined the oxygen-dependent differential activity of tirapazamine in solid tumors in vivo by using fluorescence-activated cell sorting with clonogenicity assays for cell viability or with the comet assay for DNA damage. The preferential activity of tirapazamine against hypoxic vs. aerobic tumor cells in vivo was approximately threefold, much less than the factors of 50-500 typically seen in vitro. Interestingly, we also found that tirapazamine administration often modified tumor blood flow in the murine models, an effect that could be of clinical utility in sufficiently sensitive tumor cells. Taken together, our observations suggest that sequencing of tirapazamine with other agents requires careful consideration in the clinic.

Noninvasive phosphorus-31 nuclear magnetic resonance spectroscopy (31P-NMR) was utilized to investigate the mechanism of potassium chloride (KCl) toxicity in zebra mussels. 31P-NMR spectra showed several bioenergetically important high energy phosphates, including ATP, ADP, phosphoarginine, and inorganic phosphate. Exposure of mussels to 8.6 mM KCl caused changes in these metabolites and proved lethal to the mussels when exposed over a 24 h period. This result matched that of the molluscicide Bayluscide. Here, losses in phosphoarginine and ATP were accompanied by a rise in inorganic phosphate and a shift toward intracellular acidosis. KCl levels below 8.6 mM caused marked changes in mussel high energy phosphates and pH1. However, these changes were reversible once mussels were returned to fresh standard reference water (SRW). In contrast, mussels exposed to 8.6 mM KCl under hyperbaric conditions (20 mg/L O2) survived during the 24 h KCl exposure and showed normal ATP peaks and pH1. The latter mussels, however, died when O2 supplementation was removed and mussels were transferred to SRW. Exposure to hypoxic conditions (

Development of predictive assays for measuring tumour radiosensitivity has generated much recent interest, particularly with the recognition that tumour cell survival at doses of about 2 Gy may correlate well with tumour curability. Clinical data, however, suggest that overall treatment time may be of considerable significance in radioresponsive tumours, especially for rapidly growing tumours capable of accelerated repopulation. Because neither factor can be repeatedly assessed in human tumours, we used cells growing as multicell spheroids to determine whether the initial radiation response would be predictive for multifraction exposures, or whether other factors including repopulation rate should be considered. Potential problems of hypoxia and reoxygenation were avoided by using small spheroids which had not yet developed radiobiologically hypoxic regions. Repair and redistribution dominated the responses in the first two or three exposures, with repopulation playing a minor role. As the fractionation schedule was extended, however, repopulation between fractions largely determined the number of viable cells per spheroid. We conclude that the radiation response of cells from untreated spheroids provides a general indication of net sensitivity, but that repair and redistribution produces considerable variation in radiosensitivity throughout a fractionation protocol. Ultimately, repopulation effects may dominate the multifraction response.

Interconnections of forebrain auditory and vocal control nuclei were mapped in the budgerigar using pathway tracing techniques. The anatomical results indicate four circuits by which auditory informat

Interconnections of forebrain auditory and vocal control nuclei were mapped in the budgerigar using pathway tracing techniques. The anatomical results indicate four circuits by which auditory informat

Like many tumors, multicell spheroids from Chinese hamster V79-171b fibroblasts display karyotypic heterogeneity as they enlarge. This creates significant problems for analysis of univariate DNA content histograms of cells from spheroids, and even for bivariate bromodeoxyuridine (BrdUrd)/DNA flow histograms. While attempting to routinely measure spheroid cell kinetics with the latter approach, a simple alternative that overcomes many of the inherent problems emerged. The exit rate of BrdUrd-la-belled cells from the S-phase compartment(s), determined using standard DNA analysis software, is used to define the duration of S-phase and ultimately the potential doubling time of the clonogenic cell population. This approach appears to offer several advantages over the conventional “relative movement” technique for heterogeneous samples, particularly since the subjective distinction between labelled/divided tetraploid G1 cells vs. labelled/undivided diploid G2 is no longer required. Further, the technique avoids some potential artifacts that might be expected for heterogeneous tumors where cell subpopulations proliferate with quite different cell kinetics. © 1993 Wiley-Liss, Inc.

The connectivity of a region surrounding the established thalamic auditory nuclei, n. ovoidalis (Ov) and n. semilunaris parovoidalis (SPO), was explored in the ring dove by using the anterograde tracers, Phaseolus vulgaris leucoagglutinin (PHAL) and biocytin, and the retrograde tracer, fluorogold. The Ov-SPO surround received a projection from a cell group along the interface of the auditory midbrain and the n. intercollicularis, as revealed with PHAL and biocytin, and was composed of neurons exhibiting a common morphology. These features and the presence of overlapping projections from different portions of the Ov-SPO surround suggest that this region comprises a functionally discrete area, which we term the Ov shell. Single unit recording within the shell established the existence of acoustically responsive units. Both PHAL and fluorogold labeling revealed a robust projection from the Ov shell to the caudomedial hypothalamus. Major telencephalic projections of the shell terminated within the ventral paleostriatal complex, "end-zones" of the field L, the caudomedial hyperstriatum ventrale, and regions immediately dorsal and lateral to the auditory neostriatum. Except for a portion of the shell bordering medial ovoidalis, PHAL injections into the shell also labeled fibers within the caudolateral neostriatum and along the lateral neostriatal rim. The connectivity of the Ov shell suggests that this region may integrate auditory pathways with brain regions associated with endocrine mediated behavior. In addition, the shell may constitute a source of converging input to several levels of central auditory pathways.

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.

The third instar larva, pupa, egg case and eggs of Derallus angustus are described and illustrated with line drawings. Biological and behavioral observations, based on material reared in laboratory, are included. The larvae of D. angustus and D. rudis are compared. The immatures of Derallus and Berosus are also compared from a morphological and biological point of view.

The larva, pupa and egg case of Dibolocelus ovatus are described and illustrated with line drawings. Biological observations, based on material reared in laboratory, are included.

The administration of the fluorescent DNA stain, Hoechst 33342, to mice bearing the KHT sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to functional vasculature. Different protocols of administration of Hoechst 33342 were used in order to differentiate between the presence of temporary and chronically hypoxic cells. The results show a large difference in radiosensitivity between cells close to, and distant from, functional vasculature. However, this pattern of radiosensitivity is observed only when the staining period with Hoechst 33342 is short and coincides with the period of irradiation. When the radiation treatment is temporally divorced from the staining period then the radiosensitivity and staining intensity are not related. This result can be interpreted as indicating that hypoxic cells exist within this tumour as a result of fluctuations in tumour blood flow.

Despite clear evidence that the effective penetration of the anthracycline antibiotics into experimental tumors or multicell spheroids is poor, these drugs exhibit clinical activity against a variety of solid tumors. In an attempt to understand this apparent contradiction, we used the Chinese hamster V79 spheroid system and flow cytometry techniques for intra-spheroid pharmacological studies of doxorubicin and daunomycin. Our results indicate that the slow delivery of the anthracyclines to the inner cells of spheroids is due to the rapid binding of the drug by cells in the outer layers. After exposure, the anthracyclines are retained much more effectively when cells remain in intact spheroids than when the spheroids have been dispersed, resulting in considerably more cytotoxicity in situ. This result indicates a need for considerable caution in attempting to predict the anti-tumor efficacy of drugs by using either conventional cell-culture systems, spheroids that have been disaggregated immediately post-exposure, or excision assays of tumors from experimental animals. Furthermore, our results suggest the need for a critical evaluation of the significance of the multidrug resistance (MDR) phenotype for cells surrounded by other drug-containing cells as opposed to single cells in drug-free culture medium.

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.

Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti-tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti-neoplastic drugs, it is suggested that 'drug resistance' in tumours can be a kinetic as well as a genetic problem.

The effect of cell respiration on the availability of intracellular oxygen was investigated by comparing the radiosensitivities of respiring and non-respiring cells over a range of oxygen tensions. Monolayers of Chinese hamster V79 fibroblasts on glass Petri dishes were irradiated at respiration-inhibiting (4 degrees C) and normal (37 degrees C) cell-culturing temperatures. Desired extracellular oxygen concentrations were achieved by aspirating the culture medium above the cells prior to irradiation, leaving a residual thin film which prevented drying of the cells while allowing rapid equilibration with the overlying gas. Measurement of clonogenic survival revealed that, at equivalent extracellular oxygen concentrations, the cells irradiated at 37 degrees C were less radiosensitive than those irradiated at 4 degrees C. The difference in respiring and non-respiring cell radiosensitivity was dependent on cell shape, and decreased when the cells attached to the Petri dish surface were allowed to assume a flatter configuration. These results imply that at low extracellular oxygen tensions the oxygenation of critical cellular radiation target(s) is dependent on respiration and diffusion distance, as would be expected if oxygen gradients induced by respiration exist within and immediately around actively metabolizing cells.

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-1a 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-1a 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 HIF1a expression over the course of treatment. Results: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1a, 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-1a (r = 0.31) and CAIX and HIF-1a (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-1a 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-1a, and high CAIX expression in the absence (or low levels) of HIF-1a may indicate a different

Transient hypoxia is a poorly understood and potentially important factor that may limit tumor response to various forms of therapy. We assessed transient hypoxia on a global scale in two different human tumor xenografts by sequentially administering two hypoxia markers followed by quantification of hypoxic cells using flow cytometry. High levels of the first hypoxia marker (pimonidazole) were maintained in the circulation over an 8-hour period by multiple hourly injections, providing a “time-integrated” hypoxia measure showing an asymptotic increase in the total number of hypoxic cells. Subsequent administration of a second hypoxia marker (CCI-103F) showed that substantial numbers of the previously pimonidazole-labeled cells were no longer hypoxic during the circulation lifetime of the second marker. The overall fraction of tumor cells that demonstrated changes in hypoxic status with time increased with different kinetics and by different magnitudes in the two xenograft systems. Specifically, up to 20% of the cells in SiHa (human cervical squamous cell carcinoma) tumors and up to 8% of the cells in WiDr (human colon adenocarcinoma) tumors were intermittently hypoxic over an 8-hour period. Also, the tumor cells that demonstrated transient hypoxia were typically not adjacent to functional tumor blood vessels. Similar approaches could be used in the clinic to provide information on the duration of intermittent hypoxia episodes and the fraction of transiently hypoxic tumor cells, which would, in turn, have important implications for the strategic improvement of cancer therapy.

The standard nomenclature that has been used for many telencephalic and related brainstem structures in birds is based on flawed assumptions of homology to mammals. In particular, the outdated terminology implies that most of the avian telencephalon is a hypertrophied basal ganglia, when it is now clear that most of the avian telencephalon is neurochemically, hodologically, and functionally comparable to the mammalian neocortex, claustrum, and pallial amygdala (all of which derive from the pallial sector of the developing telencephalon). Recognizing that this promotes misunderstanding of the functional organization of avian brains and their evolutionary relationship to mammalian brains, avian brain specialists began discussions to rectify this problem, culminating in the Avian Brain Nomenclature Forum held at Duke University in July 2002, which approved a new terminology for avian telencephalon and some allied brainstem cell groups. Details of this new terminology are presented here, as is a rationale for each name change and evidence for any homologies implied by the new names. Revisions for the brainstem focused on vocal control, catecholaminergic, cholinergic, and basal ganglia-related nuclei. For example, the Forum recognized that the hypoglossal nucleus had been incorrectly identified as the nucleus intermedius in the Karten and Hodos (1967) pigeon brain atlas, and what was identified as the hypoglossal nucleus in that atlas should instead be called the supraspinal nucleus. The locus ceruleus of this and other avian atlases was noted to consist of a caudal noradrenergic part homologous to the mammalian locus coeruleus and a rostral region corresponding to the mammalian A8 dopaminergic cell group. The midbrain dopaminergic cell group in birds known as the nucleus tegmenti pedunculopontinus pars compacta was recognized as homologous to the mammalian substantia nigra pars compacta and was renamed accordingly; a group of gamma-aminobutyric acid (GABA)ergic neurons at the lateral edge of this region was identified as homologous to the mammalian substantia nigra pars reticulata and was also renamed accordingly. A field of cholinergic neurons in the rostral avian hindbrain was named the nucleus pedunculopontinus tegmenti, whereas the anterior nucleus of the ansa lenticularis in the avian diencephalon was renamed the subthalamic nucleus, both for their evident mammalian homologues. For the basal (i.e., subpallial) telencephalon, the actual parts of the basal ganglia were given names reflecting their now evident homologues. For example, the lobus parolfactorius and paleostriatum augmentatum were acknowledged to make up the dorsal subdivision of the striatal part of the basal ganglia and were renamed as the medial and lateral striatum. The paleostriatum primitivum was recognized as homologous to the mammalian globus pallidus and renamed as such. Additionally, the rostroventral part of what was called the lobus parolfactorius was acknowledged as comparable to the mammalian nucleus accumbens, which, together with the olfactory tubercle, was noted to be part of the ventral striatum in birds. A ventral pallidum, a basal cholinergic cell group, and medial and lateral bed nuclei of the stria terminalis were also recognized. The dorsal (i.e., pallial) telencephalic regions that had been erroneously named to reflect presumed homology to striatal parts of mammalian basal ganglia were renamed as part of the pallium, using prefixes that retain most established abbreviations, to maintain continuity with the outdated nomenclature. We concluded, however, that one-to-one (i.e., discrete) homologies with mammals are still uncertain for most of the telencephalic pallium in birds and thus the new pallial terminology is largely devoid of assumptions of one-to-one homologies with mammals. The sectors of the hyperstriatum composing the Wulst (i.e., the hyperstriatum accessorium intermedium, and dorsale), the hyperstriatum ventrale, the neostriatum, and the archistriatum have been renamed (respectively) the hyperpallium (hypertrophied pallium), the mesopallium (middle pallium), the nidopallium (nest pallium), and the arcopallium (arched pallium). The posterior part of the archistriatum has been renamed the posterior pallial amygdala, the nucleus taeniae recognized as part of the avian amygdala, and a region inferior to the posterior paleostriatum primitivum included as a subpallial part of the avian amygdala. The names of some of the laminae and fiber tracts were also changed to reflect current understanding of the location of pallial and subpallial sectors of the avian telencephalon. Notably, the lamina medularis dorsalis has been renamed the pallial-subpallial lamina. We urge all to use this new terminology, because we believe it will promote better communication among neuroscientists. Further information is available at http://avianbrain.org

Publisher Summary DNA is the most common intracellular target for the action of agents used to treat malignant disease, and it is an essential target for mutagenic agents. The comet or single-cell gel electrophoresis method can detect, either directly or indirectly, a variety of DNA lesions, including single-strand breaks, doublestrand breaks, interstrand cross-links, and damage to the DNA bases. Apoptotic cells can be easily identified because highly fragmented DNA in apoptotic cells migrates efficiently. As few cells are required for this method, fluorescence-activated cell sorting becomes a practical way to provide purified, populations for analysis of DNA damage. Comets can be stained with antibodies against incorporated bromodeoxyuridine (BrdUrd) or against specific DNA adducts. Much of the popularity of the comet assay is due to the ability of this method to measure DNA damage in virtually any single cell. A more unique aspect of this method is the ability to detect heterogeneity in response within different cells of a single population. Under alkaline conditions, the comet assay detects DNA single-strand breaks, double-strand breaks, and alkali labile lesions.

Drug resistance is generally considered to be a major impediment to successful cancer chemotherapy, yet it is generally not possible to predict the degree or timing of the emergence of tumour resistance in most chemotherapy protocols. Recent developments with the single-cell gel electrophoresis or 'comet' assay for DNA damage at the single-cell level suggest that this technique might provide a method for identifying and potentially monitoring tumour cell responsiveness to many anti-cancer agents in situ. In principle, this assay could be applied to any accessible tumour being treated with chemotherapeutic agents that cause overt DNA damage. We have investigated that supposition using several rodent and human tumour cell lines exhibiting a spectrum of resistance to the DNA strand-breaking drug, etoposide. By assessing cells grown as monolayers, spheroids and xenografted tumours in immunodeficient mice, we found that the comet assay can provide not only an index of sensitivity to etoposide, but, additionally, can demonstrate the efficacy (or lack thereof) of multidrug resistance (MDR) reversing agents for cells in vitro, and tumours in vivo.

Budgerigars throughout life are capable of learning to produce many different sounds including those of human speech. Like humans, budgerigars use multiple craniomotor systems and coordinate both orosensory and auditory feedback in specialized forebrain nuclei. Although budgerigar auditory-vocal learning has a different evolutionary origin from that of human speech, both the human and budgerigar systems can control F0 and can alter the distribution of energy in spectral bands by adjusting the filter properties of the vocal tract. This allows budgerigars to produce an extremely diverse array of calls including many broadband and highly complex sounds.

Cells from multicellular spheroids are often more resistant than monolayers to drugs and radiation. While explanations for resistance can be based on differences in cell cycle distribution, inability of the drug to penetrate the spheroid, or the presence of hypoxic cells, these mechanisms do not adequately explain resistance to all agents. Small spheroids (containing about 25-50 cells) exposed to ionizing radiation, hyperthermia, photodynamic therapy, or topoisomerase II inhibitors, are more resistant to killing than monolayers; the close three-dimensional contact in spheroids has been implicated in this resistance. Proposed mechanisms for the 'contact effect' include gap junctional 'reciprocity', cell shape mediated changes in (repair-related) gene expression, and alterations in chromatin packaging which influence DNA repair. The consequences of the contact effect are especially important for multifraction exposures. Another form of resistance can be demonstrated during repetitive treatments; 'regrowth resistance' reflects the capacity of spheroid cells to proliferate more efficiently to compensate for cell killing.

Publisher Summary This chapter describes the calibration procedure of photomultiplier tube (PMT). The PMT calibration procedure requires ≤30 minutes for each filter-fluorochrome combination; the procedures are found to be sufficiently useful that express all quantitative measurements only in terms of calculated intensities. To facilitate these calculations further, it is useful to monitor PMT voltages and amplifier settings electronically in a manner that permits automated recording and use of these values by software packages. It should be obvious that the procedures allow standardization of fluorescence measurements, but do not substitute for the usual techniques of instrument alignment or for the necessity of internal controls in high-precision studies. These procedures do, however, permit quantization of fluorescence intensity over the entire usable range of the detectors, without analog-to-digital converter (ADC) or amplifier limitations. In view of the operational convenience of acquiring data with logarithmic amplification, the chapter also describes a method for conversion of logarithmic data to linear format.

Pathways associated with a recently defined region of the avian auditory thalamus, the shell of the nucleus ovoidalis (Ov), were examined for met-enkephalin immunoreactivity. The presence of enkephalin-like immunoreactive (ELI) perikarya within the medial margin of the inferior colliculus (ICM), afferent to the Ov shell, implicated ICM as a source of ELI fibers within the Ov shell and tract. The shell also contained ELI perikarya and its targets, including the ventromedial hypothalamus and caudoventral paleostriatal complex, were characterized by ELI fields. These data suggest that enkephalinergic auditory pathways, in parallel with traditionally recognized auditory projections, target regions of the avian basal forebrain.

Alkaline and neutral gel electrophoresis of individual mammalian cells allows detection of DNA single- and double-strand breaks, respectively. For both the alkaline and the neutral assays, lysis conditions influence how much DNA migrates, and factors in addition to DNA size play a role in migration. In particular, the tight packing of DNA in individual nuclei appears to reduce the ability to detect double-strand breaks in all of the genome. Tangling of DNA molecules is probably also responsible for the presence of "wings" associated with each nucleus after application of pulsed-field gel electrophoresis; these wings were aligned in the directions of the pulsed field, not along the resultant vector of the fields as was expected. The choice of fluorescent staining methods (propidium iodide, Hoechst 33342, or antibodies against bromodeoxyuridine) did not influence sensitivity for detecting DNA damage.

The cytotoxicity of two antineoplastic drugs, cisplatin and CCNU, was evaluated in Chinese hamster V79 multicell spheroids using the drugs as single agents or combinations. Cells obtained from different depths within spheroids 550-750 microns in diameter showed different sensitivities to the two agents; the external cells of the spheroids were more sensitive than the internal cells to cisplatin, whereas the internal cells were most effectively killed by CCNU. Combining the two agents produced the expected 'complementary' activity, and in addition, synergism was observed between the drugs at exposure levels practical for clinical use. For the combination treatments, both the net pattern of cell killing through the spheroid and the degree of interaction between the agents (quantified using the combination index method) were a function of the dose ratio of the two drugs, and of overall treatment intensity. BCNU produced patterns of cell killing similar to CCNU, but showed little interaction with cisplatin. Our results suggest significant clinical potential in using CCNU with cisplatin, particularly since CCNU-cisplatin combinations were synergistic even in the cell subpopulations most resistant to each drug as a single agent.

Publisher Summary The widespread availability of fluorescence-activated cell sorters (FACS) allows not only the quantitative demonstration of biological heterogeneity among the cells of tissues but also the ability to selectively recover cells with particular characteristics. Fluorescent probes that recognize variations in the expression of surface molecules, proteins, and DNA or RNA have become fairly commonplace; a number of functional stains have subsequently been developed that are responsive to membrane potential, respiratory activity, intracellular pH, and a growing list of other parameters. Recognition of the micro-environmental heterogeneities in growing spheroids prompted the development of techniques for the isolation of specific, defined cell subpopulations based on cellular position within the spheroid mass. Cell sorting techniques with Hoechst or carbocyanines as perfusion probes utilize the rapid binding of these agents as they diffuse from the medium into a spheroid or from the blood vessels into a tumor. Both agents remain localized in undamaged target cells after the multicell structure is disaggregated into a single-cell population. From this, it follows that sorting cells of given fluorescent intensities permits the recovery of subpopulations from the equivalently perfused regions of the spheroid or tumor system.

Publisher Summary Flow cytometers measures fluorescence intensity, but no standard method for the quantitative expression of results has been adopted. It is due to the dependence of these data on instrument alignment, laser power, optical filters, photomultiplier tube (PMT) sensitivity, and display mode and resolution. With so many factors potentially contributing to the values assigned to any sample intensity, it is often most convenient to intercompare experimental samples simply by choosing appropriate machine settings and collecting a frequency histogram of fluorescence intensity for each sample without changing the measurement conditions. This approach produces data with the greatest possible resolution when small differences in sample intensity are expected. In the case of linear amplification, the resolution and dynamic range are dependent on the number of channels selected for analog-to-digital signal conversion; for logarithmic amplification, the dynamic range is limited by that of the amplifier itself, and overall resolution is decreased. The chapter describes a method for the conversion of logarithmic data to linear format. Using this procedure, data acquired in either the log or linear format can be reduced to the same values for relative fluorescence intensity.

The TK6 and WI-L2-NS human lymphoblast cell lines are derived from the same donor, but WI-L2-NS cells are more resistant to killing by ionizing radiation and also contain a mutation in the coding region of the p53 gene which delays the appearance of apoptosis. To determine whether the difference in radiosensitivity is a result of the slower rate of appearance of apoptosis (referred to as the rate of apoptosis) or to other factors related to the function of p53 such as the G1-phase checkpoint, we have examined the clonogenicity and rates of apoptosis in synchronous populations of these two cell lines as a function of time after exposure to X rays. The greater radiosensitivity of G1-phase TK6 cells compared to G1-phase WI-L2-NS cells appeared adequate to explain the difference in radiosensitivity of asynchronous cultures. However, TK6 cells irradiated in G1 phase underwent apoptosis about three times more slowly than cells irradiated in other phases of the cycle, cautioning against equating more rapid apopt...

In earlier studies using asynchronously growing Chinese hamster cells, we observed substructure in the survival response at low doses. The substructure appeared to result from subpopulations of cells having different, cell cycle phase-dependent radiosensitivity. We have now applied the same flow cytometry and cell sorting technique to accurately measure the responses of cells of eight different asynchronously growing human tumor cell lines, representing a wide range in radiosensitivity. When the data were fitted with a linear-quadratic (LQ) function, most of these lines showed substructure similar to that observed in Chinese hamster cells, with the result that values of α and β were dependent on the dose range used for fitting. Values of α describing the low-dose response were typically smaller (by as much as 2.2 times) than the α describing the high-dose response, while values of β were larger at low doses. Values of α/β from our measurements are in reasonable agreement with other values published recent...

Measurements of relative biological effectiveness (RBE) have been made on the range-modulated 70 MeV proton beam at TRIUMF using a precise cell sorting survival assay. In this study, Chinese hamster V79-WNRE cells were suspended in medium containing liquid gelatin at 37 degrees C in irradiation tubes and the gel was allowed to solidify by cooling to 4 degrees C. Complete cell survival responses were measured at 11 positions with 2 mm spacing within a proton stopping peak width of approximately 2 cm. Survival responses after proton irradiation were compared with responses to 60Co gamma rays measured at the same time, and RBE values were determined as a function of both dose and depth. Above doses of 4 Gy, the average RBE for these cells throughout the modulated proton stopping distribution was 1.21 +/- 0.05, measured at a survival of 1%. However, we also observed that, within the spread-out Bragg peak, the RBE increased with increasing depth, from approximately 1.2 at the proximal part to > 1.3 at the distal part of the peak. At the distal edge of the stopping distribution, the RBE value increased significantly, to an extent that may be of concern when this region of the treatment volume is close to sensitive tissues. Below 4 Gy, the RBE value was also dependent on radiation dose, increasing significantly to values of approximately 1.37 and 1.56 at 2 and 1 Gy, respectively. Our results illustrate that the use of a single RBE value in different irradiation protocols can be an oversimplification, and argues for the use of "proton gray doses" rather than "gamma-ray equivalent grays."

In an earlier study using cell sorting techniques to define the radiation survival response of asynchronous Chinese hamster V79-171 cells more accurately, we found evidence of substructure in the response at low dose. In the present work we have attempted to show that this substructure arises from the subpopulations of sensitive (G1, G2 phase) and resistant (late S phase) cells which are present in asynchronously dividing cultures but which are not resolved by conventional survival assays. Partially synchronized cells were produced by exposure to 1 mM hydroxyurea for 12 h and were harvested 15 min later, yielding a population of viable cells at or just beyond the G1/S-phase boundary. Parallel experiments were carried out with asynchronous cells. The average of repeated measurements of the radiation survival response of asynchronous cells again showed a significant difference (P = 0.002 to 0.009) between the alpha and beta values evaluated from the data for the low-dose range, 0-2.8 Gy, and the high-dose range, 2.8-14 Gy. For G1/S-phase cells, however, there was no significant difference between the values of alpha and beta for the low-dose and high-dose ranges (P > 0.5). The results thus support the hypothesis that the observed substructure in the asynchronous response is due to resolution of subpopulations of different radiosensitivities, and they illustrate the advantage of the cell sorter assay for accurate measurements of cell survival, particularly at low dose.

Flow cytometry and cell sorting techniques have been used together with repeated measurement in an attempt to define better the radiation survival response of asynchronously dividing Chinese hamster V79-171 cells under aerobic and hypoxic conditions. Although the first two decades of cell inactivation have been examined, particular attention has been given to the low-dose range of a few grays, as used in individual radiation therapy treatments. A single linear-quadratic dose-response function was consistently unable to fit both the low-dose and high-dose data satisfactorily, suggesting a two-component response. Separate fitting of the low-dose and high-dose portions of the response yielded alpha and beta values which differed significantly (P = 0.001 to 0.002). The data are consistent with the hypothesis that the observed substructure simply reflects the presence of subpopulations of sensitive (G1-, G2-phase) and resistant (late S-phase) cells, which are resolved in these measurements. These results may have significance for certain situations in radiation therapy and in biophysical modeling of the radiation response.

Some of the biological changes associated with extreme hypoxia at 37 degrees C (less than 10 ppm pO2) were examined in Chinese hamster V79 cells. Specifically, extreme hypoxia caused an initial decrease in plating efficiency to 55% in 4 hr after the onset of hypoxia. Beyond this time, the decline in plating efficiency was more gradual reaching 35% of control at 20 hr. Flow microfluorimetry (FMF) studies, in which cells are sorted on the basis of DNA content and then assayed for viability, demonstrated that mid S phase cells were most sensitive to chronic hypoxia, with surviving fraction equal to 2.5% at 20 hr. Furthermore, the viability of G1 and G2/M cells, after 20 hr of hypoxic storage, was also reduced to 20 and 7.6%, respectively. Hypoxia also caused alterations in the cell cycle distribution of initially asynchronous cells, as determined by dual parameter FMF measurements of both cellular DNA content and incorporated BudR. In particular, G2/M cells completed mitosis, while G1 cells showed little or no movement. Lastly, cells stored in chronic hypoxia displayed an enhanced radiosensitivity when compared to acutely hypoxic cells. Possible reasons for these observations are discussed.

Chinese hamster V-79 cells, when grown in suspension culture as multicell spheroids, display many of the features of organized tissue. AF-2, a known mutagen, was used to examine the influence of some of these factors on mutagenesis. Since AF-2 is fluorescent, FCM quantitation of intracellular drug content was performed after incubation of spheroids under either aerobic or hypoxic conditions. For the same level of toxicity, hypoxic spheroids accumulated twice as much AF-2 as aerobic spheroids, but showed less than half the number of mutants resistant to 6-thioguanine. Using velocity sedimentation techniques, a selective increase in the toxic and mutagenic effects of AF-2 was found for internal vs external cells of the spheroids. However, in mutagenicity experiments using all the cells from spheroids, the response of the external cycling cells predominated over the response of the internal cells. Thus, in addition to the demonstration that cellular environment can modify mutagenesis, these results also suggest that the growth fraction of the target cells can be an important consideration in mutation experiments.