An open-source deconvolution software package for 3-D quantitative fluorescence microscopy imaging.

Deconvolution techniques have been widely used for restoring the 3-D quantitative information of an unknown specimen observed using a wide-field fluorescence microscope. Deconv, an open-source deconvolution software package, was developed for 3-D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3-D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.

CELL SEGMENTATION USING HESSIAN-BASED DETECTION AND CONTOUR EVOLUTION WITH DIRECTIONAL DERIVATIVES.

The large amount of data produced by biological live cell imaging studies of cell behavior requires accurate automated cell segmentation algorithms for rapid, unbiased and reproducible scientific analysis. This paper presents a new approach to obtain precise boundaries of cells with complex shapes using ridge measures for initial detection and a modified geodesic active contour for curve evolution that exploits the halo effect present in phase-contrast microscopy. The level set contour evolution is controlled by a novel spatially adaptive stopping function based on the intensity profile perpendicular to the evolving front. The proposed approach is tested on human cancer cell images from LSDCAS and achieves high accuracy even in complex environments.

Segregation of genomes in polyploid tumour cells following mitotic catastrophe.

Following irradiation p53-function-deficient tumour cells undergo mitotic catastrophe and form endopolyploid cells. A small proportion of these segregates nuclei, and give rise to viable descendants. Here we studied this process in five tumour cell lines. After mitotic failure, tumour cells enter the endocycle and form mono-nucleated or multi-nucleated giant cells (MOGC and MNGC). MNGC arise from arrested anaphases, MOGC, from arrested metaphases. In both cases the individual genomes establish a radial pattern by links to a single microtubule organizing centre. Segregation of genomes is also ordered. MNGC present features of mitosis being resumed from late anaphase. In MOGC the sub-nuclei retain arrangement of stacked metaphase plates and are separated by folds of the nuclear envelope. Mitosis then resumes in sub-nuclei directly from metaphase. The data presented indicate that endopolyploid tumour cells preserve the integrity of individual genomes and can potentially re-initiate mitosis from the point at which it was interrupted.

Development of the large scale digital cell analysis system.

Quantitative analysis of large numbers of cells exposed to various stresses would be facilitated by an automated microscope system designed to monitor up to 1000 microscope fields over time intervals of up to one month. The Large Scale Digital Cell Analysis System (LSDCAS) was designed by the authors to provide such analysis capabilities. This report presents a description of the development of the LSDCAS and its application to the study of cells undergoing radiation-induced mitotic catastrophe. A detailed description of the data acquisition algorithms used and the hardware configuration underlying the system is presented. Quantitative analysis of colony formation of cells exposed to 5 Gy X irradiation is presented. Upon analysis, it was found that the plating efficiency in irradiated populations agreed with that obtained using conventional colony-formation assays, indicating the accuracy of LSDCAS in the determination of cell clonogenicity. An unexpected finding was that fully 50% of the surviving colonies exhibited mitotic catastrophe that persisted throughout colony formation. Ongoing extension of the capabilities of the LSDCAS using image segmentation techniques applied to a variety of cellular endpoints is then briefly described.

Enhancement of radiation-induced mitotic catastrophe by moderate hyperthermia.

PURPOSE: To determine if a maximally radiosensitizing but non-toxic moderate hyperthermia treatment enhances radiation-induced mitotic catastrophe. MATERIALS AND METHODS: HeLa S3 cells were given a non-lethal heat treatment (41.5 degrees C, 4 h) and irradiated with 5 Gy X-irradiation. Alterations in cell-cycle distribution, intracellular cyclin B1 levels, and the yield of mitotic catastrophe were then measured and compared to heat-only and radiation-only groups, as a function of time following treatment. RESULTS: A greater accumulation of cells in S and G2 phases of the first cell-cycle post-treatment was observed in the combined heat and radiation groups, when compared with that observed following treatment with heat or radiation alone. Similarly, intracellular levels of cyclin B1 and the incidence of mitotic catastrophe were found to be greater in the combined treatment groups. CONCLUSIONS: This study provides further evidence that delays late in the cell cycle are implicated in increases in intracellular cyclin B1 levels and the subsequent development of mitotic catastrophe. Further, these data suggest a role for mitotic catastrophe occurring as a result of G2/M checkpoint abrogation in the process of thermal radiosensitization.

Mitotic catastrophe induced by exposure of V79 Chinese hamster cells to low-energy protons.

PURPOSE: To determine the yield of mitotic catastrophe induced after low energy proton irradiation and to compare this yield with that induced by X-rays. MATERIALS: Asynchronous Chinese hamster V79 cells were irradiated with 0.5, 1, 2, 5 and 10 Gy proton beams and X-rays. Proton LET of 7.7, 11.0 and 30.5 keV microm(-1), corresponding to energies of 5.01, 3.20 and 0.76 MeV respectively, evaluated at the cell mid-plane, were used for experiments. The occurrence and yield of mitotic catastrophe was measured as the percentage of cells exhibiting fragmented nuclei. RESULTS: Proton irradiation led to an enhanced induction of mitotic catastrophe in V79 cells. The onset of nuclear fragmentation, a hallmark of mitotic catastrophe, occurred much earlier after cell exposure to proton particles than to X-ray irradiation. CONCLUSIONS: Mitotic catastrophe is persistent in the subsequent cell generations after proton and X irradiation of V79 cells; but protons are more effective than X-rays for the induction of this phenomenon. These results are discussed in terms of their importance in space exposures and possible acquisition of genomic instability by the progeny of irradiated cells.

Delayed DNA damage associated with mitotic catastrophe following X-irradiation of HeLa S3 cells.

Partial loss of the radiation G2/M checkpoint is thought to be an early event in cell immortalization. One of the attributes of immortalized cell lines is an increase in susceptibility to induction of genomic instability by clastogenic agents. Recently we have shown that in irradiated HeLa cells cell cycle delays in late S and G2 lead to overaccumulation of cyclin B1 and that enhanced intracellular levels of this positive regulator of the cell cycle is correlated with cyclin-dependent kinase activation, spontaneous premature chromosome condensation and subsequent mitotic catastrophe occurring following irradiation. Previous studies have shown that spontaneous premature chromosome condensation and mitotic catastrophe are independent of apoptosis. This report shows that 40 h following X-irradiation of HeLa S3 cells, and subsequent to mitotic catastrophe, DNA strand breaks appear which are chemically distinct from those initially produced by ionizing radiation. This delayed damage is recognized by terminal transferase and thus involves generation of free 3'-OH ends. Pulse field gel electrophoresis analysis of DNA size distributions shows that DNA fragments of approximately 40 kbp and smaller are produced. As strand breaks produced as a direct result of irradiation are generally repaired within a few hours after exposure to X-rays at the doses used, these results describe a novel mechanism for generation of DNA damage occurring a day or more following irradiation. These results may be pertinent to the understanding of mechanisms underlying the delayed lethal effects of irradiation and may provide an initiating mechanism for radiation-induced genomic instability.

An analysis of testosterone implants for androgen replacement therapy.

OBJECTIVE: To review 13 years of experience using fused crystalline testosterone implants for androgen replacement therapy in order to identify pattern of usage (including continuation rates) and adverse events emerging during therapy and factors associated with adverse events including implant extrusions. DESIGN: Retrospective review of prospectively collected data on characteristics of patients and implant procedures performed as well as adverse events reported during routine follow-up. PATIENTS: Over 13 years 973 implant procedures using fused crystalline testosterone implants were performed in 221 men. MEASUREMENTS: Continuation rates and adverse events such as extrusions, bleeding, infection or others were recorded and analysed in relationship to characteristics of the patient and the implant procedure performed. RESULTS: Overall rate of adverse events (108/73, 11.1%) was significantly related to increased numbers of implants (4.2 +/- 0.1 vs 4.0 +/- 0.03, P = 0.031) and higher levels of physical activity at work (P = 0.030). The most common adverse effect was extrusion (83/973, 8.5%) which was related to occupational classification (P = 0.033) and increasing work activity (P = 0.044) and occurred more frequently than by chance in multiple (16 vs 3.3 expected) rather than single (65 vs 76.1 expected) episodes. Bleeding (22/973, 2.3%) was significantly associated with an increased number of implants (4.5 +/- 0.2 vs 4.0 +/- 0.03, P = 0.020) but even in the worst cases (3/22) it was of minor clinical importance. Infection was rare (6/973, 0.6%) but occurred more among thinner men. The overall continuation rate was 92.7% increasing from 86% after the first implantation to > 99% after the tenth implant. CONCLUSIONS: This study demonstrates the very satisfactory clinical acceptability of testosterone pellet implants for androgen replacement therapy within a single unit with experienced operators. The only regular adverse effect is extrusion, which may be related to mechanical factors such as habitual work activity but also possibly procedural factors. Other adverse effects such as bleeding, infection and fibrosis were rare. An improved method of implant delivery would enhance this old but durable technology.

Spontaneous premature chromosome condensation and mitotic catastrophe following irradiation of HeLa S3 cells.

PURPOSE: To study the mechanisms underlying the loss of G2/M checkpoint control which leads to mitotic catastrophe in human tumour cells following exposure to ionizing radiation. MATERIALS AND METHODS: Asynchronous HeLa S3 cells were irradiated with doses of 5, 10 and 20 Gy X-rays. Cell-cycle progression and cyclin B1 levels were measured using bivariate flow-cytometric techniques as a function of time after irradiation. As indicators of mitotic catastrophe, the appearance of spontaneous premature chromosome condensation (SPCC) and cells presenting nuclear fragmentation were analysed using microscopy. Cyclin B1-dependent kinase activity was determined in immunoprecipitates and analysed using gel electrophoresis. RESULTS: After X-irradiation of HeLa cells, delays in late S and G2 phases of the cell cycle were followed by SPCC and nuclear fragmentation, both indicative of mitotic catastrophe. The kinetics of appearance of cells that had apparently undergone mitotic catastrophe (i.e. the fraction of cells exhibiting nuclear fragmentation) was independent of the dose-dependent radiation-induced division delay, while the extent of fragmentation (expressed as the number of nuclear fragments per fragmented cell) did increase with dose. Also observed was a 5-fold elevation of cyclin B1 levels in late S/G2 cells, which correlated temporally with the observed delays late in the cell cycle. Following the appearance of elevated cyclin levels, cyclin B1-associated histone H1 kinase activity showed similar increases; these increases in kinase activity occurred prior to increases in the fraction of cells exhibiting nuclear fragmentation. CONCLUSIONS: In human cells, cyclin B1 gene expression occurs in late S and G2 phases, and thus the increase observed in this protein may be due to the increased time spent by cells in these phases as a result of cell-cycle delays caused by the radiation exposure. It is possible that, under these conditions, over accumulation of cyclin B1 dilutes the mitosis-inhibitory action of the weel or other inhibitory pathways. Thus, this study presents a possible mechanism for G2/M checkpoint abrogation following ionizing radiation which may depend solely on effects associated with perturbed cell-cycle progression.

Establishing the minimum effective dose and additive effects of depot progestin in suppression of human spermatogenesis by a testosterone depot.

Hormonally induced azoospermia induced by weekly im injections of testosterone enanthate provides effective and reversible male contraception, but more practical regimens are needed. Given our previous findings that six 200-mg pellets implanted subdermally produced more stable, physiological T levels and reduced the delivered T dose by more than 50% while maintaining equally effective suppression of sperm output with fewer metabolic side-effects than weekly 200-mg testosterone enanthate injections, we sought in this study to determine 1) whether further dose-sparing could be achieved by lower testosterone doses while maintaining efficacy and 2) the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose as a model depot progestin/androgen combination male contraceptive. Healthy volunteers were randomized into groups (n = 10) who received either of two lower T doses (two or four 200-mg T pellets) or four 200-mg T pellets plus a single im injection of 300 mg depot medroxyprogesterone acetate (DMPA). Two T pellets (400 mg, 3 mg/day) had a negligible effect on sperm output. Four T pellets (800 mg, 6 mg/day) suppressed sperm output between the second to fourth postimplant months; output returned to normal by the seventh postimplant month, although only 4 of 10 men became azoospermic or severely oligozoospermic (< 3 mol/L/mL). The addition of a depot progestin markedly increased the extent, but not the rate, of sperm output suppression, with 9 of 10 becoming azoospermic and 10 of 10 becoming severely oligozoospermic. There were no serious adverse effects during the study. Plasma total and free testosterone levels remained within the eugonadal range at all times with each treatment. Plasma epitestosterone was suppressed by all 3 regimens, consistent with a dose-dependent inhibition of endogenous Leydig cell steroidogenesis. Plasma LH and FSH measured by a two-site immunoassay were suppressed in a dose-dependent fashion by T and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either T dose. Prostate-specific antigen and lipids (total, low or high density lipoprotein cholesterol, and triglycerides) were not significantly changed in any group. Thus, a depot testosterone preparation with zero order release must be delivered at between 6-9 mg/day to provide optimal (but not uniform) efficacy at inducing azoospermia. The addition of a single depot dose of a progestin to a suboptimal testosterone dose (6 mg/day) markedly enhances the extent, but not the rate, of spermatogenic suppression, with negligible biochemical androgenic side-effects. These findings provide a basis for the use of a progestin/androgen combination depot for hormonal male contraception.

PIP: Clinical research conducted in Australia suggests that a progestin-androgen combination depot has potential for hormonal male contraception. The authors' previous research had indicated that 6 200-mg testosterone enanthate pellets implanted subdermally produced substantial reductions over injections in the delivered testosterone dose while maintaining equally effective suppression of spermatogenesis with few metabolic side effects. The present study sought to determine whether lower testosterone doses would maintain efficiency and to assess the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose (6 mg/day). 10 volunteers received either 2 or 4 200-mg testosterone pellets or 4 200-mg pellets plus a single intramuscular injection of 300-mg depot medroxyprogesterone acetate (DMPA). The testosterone implants alone achieved inadequate suppression of spermatogenesis for a male contraceptive; 400 mg of testosterone (3 mg/day) had a negligible effect on sperm output, while 800 mg (6 mg/day) produced azoospermia or severe oligozoospermia in only 4 of 10 men. However, the addition of DMPA markedly increased the extent, but not the rate, of sperm output suppression: azoospermia was achieved in 9 men and oligozoospermia in all 10 subjects, and sperm suppression persisted for 3 months. Epitestosterone concentrations, used as a marker of Leydig cell steroidogenesis, were decreased in a time- and dose-dependent manner, reaching castrate levels in the combined group. Plasma luteinizing hormone and follicle-stimulating hormone levels were suppressed in a dose-dependent fashion by testosterone and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either testosterone dose. Prostate-specific antigens and lipids were not significantly altered by any regimen. There were no discontinuations or reports of side effects.

Uncoupling of M-phase kinase activation from the completion of S-phase by heat shock.

Chronic exposure of asynchronous HeLa cell cultures to 41.5 degrees C leads to an accumulation of cells in the S-phase, spontaneous premature chromosome condensation, and loss of clonogenicity (M.A. Mackey, S. L. Anolik, and J. L. Roti Roti. Cancer Res., 52: 1101-1106, 1992). In this report, we show that increases in histone H1 kinase activity during 41.5 degrees C exposure occur coincidentally with the appearance of premature chromosome condensation. Furthermore, this kinase activity is shown to be associated with M-phase kinase complexes containing cyclin B1. These increases in the activity of M-phase kinase were found to occur concomitantly with an elevation in cyclin B1 mRNA and an accumulation of cyclin B1 protein. Because cyclin B1 transcription begins in the S-phase, it is probable that the heat-induced delay in the S-phase allows the accumulation of abnormally high cyclin B1 levels. Elevated cyclin B1 levels could then account for the observed abrogation of the cell cycle checkpoint, which usually assures that mitosis does not proceed until DNA replication is complete. This involvement of M-phase kinase in heat-induced cytotoxicity demonstrates the importance of the coordinate regulation of the processes of DNA replication and entry into mitosis.

Spontaneous premature chromosome condensation, micronucleus formation, and non-apoptotic cell death in heated HeLa S3 cells. Ultrastructural observations.

Hyperthermia is an efficient means of inducing cell death in vivo and in vitro. Among human neoplastic cells, HeLa S3 cells are susceptible to heat injury when exposed to long duration moderate hyperthermia (41.5 C), conditions that are reproducible and sustainable in the clinical setting. Hence, HeLa S3 cells are a useful substrate for evaluation of hyperthermic injury in human neoplasia. Previous studies have demonstrated a consistent response of HeLa S3 cells to moderate hyperthermia: spontaneous premature condensation of chromosomes during heat exposure in S phase followed by apparent nuclear fragmentation and, inevitably, cell death. To further characterize the morphological features of this process, HeLa S3 cells grown in suspension at 37 C were heated for 4, 8, 12, or 16 hours at 41.5 C and harvested in glutaraldehyde for electron microscopic evaluation. Compared with untreated controls, heated samples exhibited a characteristic pattern of chromosome condensation that mimicked mitotic prophase but was followed by haphazard asymmetric segregation of chromatid clusters in abnormal metaphase/anaphase and premature reformation of nuclear membrane, resulting not in nuclear fragmentation, but in multiple micronuclei. This pattern of nuclear morphology was not observed in controls. The fraction of cells with micronuclear morphology increased with time in heated samples (from 3.6% at 4 hours to 16.6% at 16 hours), consistent with previous light microscopic analyses of nuclear fragmentation. Cells with multiple micronuclei subsequently exhibited features similar to necrotic cell death. Apoptosis was never observed. Moderate hyperthermia appears to induce a novel morphological pattern of cell injury and death in HeLa S3 cell lines that may be useful as a means of screening cell lines for nonmorphological analyses of hyperthermic injury.

Tolerability of intramuscular injections of testosterone ester in oil vehicle.

We undertook a prospective survey of the tolerability of deep i.m. injections of testosterone enanthate in a castor oil vehicle, the most widely used form of androgen replacement therapy. Over a period of 8 months, 26 men received 551 weekly injections into the gluteal, deltoid or thigh muscle and side-effects were recorded immediately and 1 week after each injection by the same nurse using a standardized questionnaire. Most injections caused no complaints [389/551, 70.6% (95% confidence interval 66.6-74.4%)] but minor local side-effects, mostly pain and bleeding, were common [162/551, 29.4% (25.6-33.4%)]; no serious side-effects were observed. Considering all side-effects, the gluteal site had fewer complaints and was less prone to bleeding but was painful more often than deltoid or thigh injection sites. The laterality of injection at any site had no significant effect on side-effects. The only systemic side-effect was episodes of sudden-onset, non-productive cough associated with faintness following eight injections [1.5% (0.6-2.9%)] which we speculate may have been due to pulmonary oil microembolism. We conclude that, when administered by an experienced nurse, deep i.m. injection of testosterone enanthate in a castor oil vehicle is generally safe and well tolerated but causes relatively frequent minor side-effects, including pain and bleeding. An improved depot form of testosterone would be highly desirable for androgen replacement therapy and hormonal male contraception.

Intense sweeteners use and weight change among women: a critique of the Stellman and Garfinkel study.

OBJECTIVE: The purpose of this study was to evaluate the work of Stellman and Garfinkel who speculated, based on epidemiologic data, that users of intense sweeteners are more likely than nonusers to gain weight. METHODOLOGY: We analyzed the study's design and statistical analysis to determine whether the conclusions of Stellman and Garfinkel were supported by the data. RESULTS: Several methodological flaws and inappropriate statistical analyses were identified. These included: use of data from an unrelated study for which they were not intended; failure to correct for bias due to convenience sampling; use of data from a subpopulation without validation; and stratification of subjects by outcome data. CONCLUSION: Our analysis indicates that the data from the study in question do not allow one to draw conclusions about a relationship between use of intense sweeteners and weight change. Furthermore, data from well-designed clinical trials have shown that aspartame is not associated with weight gain, and when used as part of a balanced deficit diet, can facilitate weight loss.

A model of heat-induced clonogenic cell death.

When cells are exposed to elevated temperatures, clonogenicity decreases in a temperature-dependent manner displaying non-linear kinetics. In this report we show that the survival of synchronized cell populations after heat exposure can be characterized by a parameter epsilon, which is normally-distributed throughout the population. Cells whose corresponding epsilon-value lies below an arbitrary threshold value epsilon min are unable to form a colony. Upon transfer of a cell culture to an elevated temperature, the mean value of the epsilon distribution decreases exponentially over time with a rate dependent upon the difference between the current and final mean value, until reaching a final value epsilon f dependent on the particular temperature used, thus representing a larger proportion of cells with epsilon less than epsilon min which are non-clonogenic. The analyses evaluate a temperature-independent parameter k which is presumed to be dependent upon the growth conditions of the cell population, and a temperature-dependent parameter epsilon f, which is characteristic of the new temperature-perturbed steady state. Comparison of the results of analysis of synchronized G1 cell survival data to that for S phase cells obtained over a range of temperatures shows that it is sufficient to ascribe the increased thermal sensitivity of S phase cells to a different k value, the temperature-dependence of epsilon f being identical in the two cases. Linear regression analysis of the temperature-dependence of epsilon f, when expressed as 1n (-epsilon f) = a0 + a1/T demonstrated strong linearity (r2 = 0.99) for either the individual (G1 or S) or combined data sets. This description of heat-induced cytotoxicity may be of use as the basis for a dose concept for the clinical administration of hyperthermia.

Cellular mechanisms associated with the lack of chronic thermotolerance expression in HeLa S3 cells.

Chronic thermotolerance is an operational definition for that resistance to cell killing by heat which develops during a protracted exposure at temperatures generally in the range of 41.5-42.5 degrees C which is usually observed as a reduction in the slope of the survival curve. While Chinese hamster ovary (CHO) cells are generally more sensitive to high-temperature heat shock than HeLa cells, studies of cells maintained in suspension culture at 41.5 degrees C demonstrated CHO cells to be more resistant to cell killing at this temperature than HeLa cells, due to the expression of chronic thermotolerance in the hamster cell line and the corresponding lack of chronic thermotolerance expression in the HeLa cell line. Experiments were conducted in the two cell lines while heating under identical conditions, in order to detect any cell line-specific changes in heat-induced perturbation of cell cycle progression and the expression of chronic thermotolerance. Our results showed that CHO cells exhibited a G1 block which lasted throughout the course of the 32-h heating period. HeLa cells, however, failed to accumulate in G1, progressing instead into S phase where spontaneous premature chromosome condensation and nuclear fragmentation were observed. This accumulation of cells with condensed chromatin possessing S-phase DNA content exhibited a linear, one-to-one functional relationship with the fraction of dead cells. Previous studies (M.A. Mackey and W.C. Dewey, Int. J. Hyperthermia, 5:405-415, 1989) demonstrated that synchronized S-phase CHO cells heated at 41.5 degrees C and 42 degrees C were unable to express chronic thermotolerance. Therefore, we hypothesize that progression of cells out of G1 phase into S and G2-M phases leads to lethal processes that prevent the expression of chronic thermotolerance in the HeLa cell line. This hypothesis is strengthened by the observed correlation between the accumulation of "mitotic-like" cells and decreased survival, suggesting that the G1 block observed in CHO cells is causally connected with the expression of chronic thermotolerance.

Changes in heat and radiation sensitivity during long duration, moderate hyperthermia in HeLa S3 cells.

Step-up heating and thermal radiosensitization were studied at 41.5 degrees C in HeLa S3 cells under conditions where chronic thermotolerance was not expressed. In spite of this lack of thermotolerance expression, it was possible that thermotolerance to higher temperature treatment had developed. Accordingly, cells were incubated for various times at 41.5 degrees C, then immediately shifted up to 45 degrees C, whereupon heating continued for up to 75 min. Thermotolerance to 45 degrees C heating was observed after 8 hr incubation at 41.5 degrees C and decayed by 32 hr of continuous incubation at 41.5 degrees C. When the time of 45 degrees C treatment was extended to 150 min, the biphasic survival response indicated that chronic thermotolerance was expressed at 45 degrees C, even though it was not expressed during the 41.5 degrees C treatment. Thus, chronic thermotolerance can develop under conditions (e.g., at 41.5 degrees C) where it is not expressed, yet be expressed under other conditions (e.g., during 45 degrees C exposure). When cultures were x-irradiated after various periods of 41.5 degrees C treatment, maximum thermal radiosensitization was observed after 4 hr of incubation at 41.5 degrees C, for which no cell killing was observed due to heat alone. The radiosensitization observed decreased the Do and Dq values from about 1.3 Gy to 0.7 Gy and from about 2.0 Gy to 1.0 Gy, respectively. As the duration of the 41.5 degrees C pre-treatment was extended up to 32 hr, no additional thermal-radiosensitization was observed; all killing due to the heat exposure at 41.5 degrees C was additive to the radiation killing after the initial induction of thermal radiosensitization. These results demonstrate differences in the thermal and radiation responses of HeLa cells when compared to earlier studies using CHO cells and may be more relevant to the clinical setting.