Effect of serum on heat response of synchronized mouse neuroblastoma cells: protection of cell cycle progression, protein synthesis and survival

The effect of serum and temperature elevation on proliferation has been studied in synchronized mouse neuroblastoma (Neuro-2A) cells. The effects of serum were studied on the induction of (a) mitotic delay due to a non-lethal heat treatment (30 min at 42.7 degrees C) and (b) the loss of colony-forming capacity after a more extensive heat treatment (45 min at 44 degrees C or a continuous 42.7 degrees C heat treatment). The following results were obtained. Under conditions of serum depletion, cell cycle extension of heated G1 phase cells was more than that of heated G2 phase cells. Serum protected against heat-induced alterations of cell cycle progression in G1- but not in G2 phase cells. This effect of serum could be mimicked by a supplement to the medium of human transferrin, bovine pancreas insulin and selenium, and was correlated with protection of protein synthesis. Serum also affected heat-induced cell killing. Under conditions of serum depletion, G1 phase cells were more resistant to heat compared to G2 cells. The presence of serum during heat treatment further increased the thermoresistance of G1 phase cells, but did not affect sensitivity of G2 phase cells. This effect of serum could not be mimicked by a supplement of transferrin, insulin and selenium. These results indicate that serum protects G1 phase cells for heat-induced changes of cell cycle progression as well as on cell survival, but the mechanisms involved in both phenomena seem to be different.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.     

Growth characteristics of human melanoma multicellular spheroids in liquid-overlay culture: comparisons with the parent tumour xenografts

The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 X 10(7) microns 3 (a diameter of 270-340 microns) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 microns. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 +/- 0.2 to 2.7 +/- 0.4 days and the fraction of cells in S from 13 +/- 1 to 28 +/- 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G1. The spheroid volume-doubling times did not correlate with those of the parent xenografts (Td = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G1/G0, S and G2 + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts--i.e. 50-80% (G1/G0), 10-30% (S) and 10-20% (G2 + M).     

The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice

A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors.     

Radiation cell survival and growth delay studies in multicellular spheroids of small-cell lung carcinoma

The radiation sensitivity of two small-cell lung carcinoma cell lines growing as multicellular spheroids in static culture was determined using clonogenic cell survival and growth delay as endpoints. Growth delay determination suggested that clonogenic cell kill was less than was obtained by direct assay of cell survival. Recovery from potentially lethal damage was assayed in one line (HC12) but was not demonstrable, and clonogenic cell survival decreased with time in treated spheroids with diameters greater than 300 microns which contained a hypoxic cell population. Microscopic examination of the treated spheroids showed the emergence of an abnormal giant-cell population, and the progressive clonogenic cell loss that occurred after treatment was thought to be due to oxygen and nutrient deprivation of the remaining viable cells by this doomed cell population. Correction of the growth delay measurements for changes in cell size and clonogenic cell population allowed correlation of the growth delay and cell survival data.     

The cell cycle dependence of thermotolerance. I. CHO cells heated at 42 degrees C

We examined the dependence of heat killing and thermotolerance on the position and progression of Chinese hamster ovary (CHO) cells in the cell cycle. We measured cell cycle perturbations and survival of asynchronous and synchronized G1-, S-, and G2-phase cells resulting from continuous heating at 42.0 degrees C for up to 80 hr. Thermotolerance under these conditions was transient in nature, was dependent on the position of cells in the cell cycle, and occurred concurrently with a heat-induced delay of progression of G1- and G2-phase cells. When G1 cells were heated, survival decreased to 25% after 4 hr, at which time the thermotolerance was expressed. For G2 cells survival decreased initially at the same rate (T0 congruent to 3 hr) but thermotolerance was not expressed until approximately 12 hr, at which time the survival was 4%. The rate of decrease in survival was much more rapid for cells heated in mid-S phase (T0 congruent to 0.5 hr), and these cells did not express thermotolerance at a measurable level. Concurrent with the expression of thermotolerance, the progression of cells heated in G1 and G2 was delayed. Following the expression of tolerance, progression resumed at a rate approximately equal to the rate of decrease in survival of the G1 population. Cells heated in mid-S phase continued to progress through the cell cycle until they reached G2, where they were also delayed.     

Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts

Survival after H2O2 exposure or heat shock of asynchronous Chinese hamster ovary cells (HA-1) was assayed following pretreatment with mildly toxic doses of either H2O2 or hyperthermia. H2O2 cytotoxicity at 37 degrees C, expressed as a function of mM H2O2 was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H2O2 as a cytotoxic agent. When the survival data were plotted as a function of mumoles H2O2/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3-5 mumoles/cell X 10(-7)) H2O2 for 1 hr at 37 degrees C (30-50% survival) became resistant to a subsequent H2O2 treatment 16-36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4-6]. Their resistance to 43 degrees C heating, however, was only slightly increased over controls 16-36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one-dimensional SDS-polyacrylamide gel was enhanced twofold in the H2O2-pretreated cells. When the cells were heated for 15 min at 45 degrees C (40-60% survival), the survivors became extremely resistant to 43 degrees C heating and somewhat resistant to H2O2 (DMF at 1% isosurvival = 2). The heat-induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat-induced resistance to H2O2 (16-36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H2O2 pretreatment. The cells that became resistant to H2O2 by either pretreatment (H2O2 or heat shock) also increased their ability to reduce the H2O2 cytotoxicity from the treatment medium beyond that of the untreated HA-1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H2O2 following mild pretreatment with H2O2 or heat shock. The cross-resistance induced by H2O2 and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H2O2 pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.     

Implications of nuclear diameter in small cell lung carcinoma

The nuclear diameter of 5,117 malignant cells from 42 small cell lung carcinoma (SCLC) patients was assessed either on pretreatment tissue sections (35 cases) or cytologic smears (7 cases) by ocular micrometry. The SCLCs were subtyped as 30 oat cell carcinomas and 12 intermediate cell carcinomas according to the World Health Organization classification, based on the predominant histology of the tumor. The median number of nuclei measured from each patient was 110. All patients were treated identically by sequential hemibody and local irradiation combined with chemotherapy and had a median follow-up time of 310 days. The mean nuclear diameter (+/- standard error) obtained from tissue sections was 8.2 +/- 0.03 microns (median = 8.0), including 7.3 +/- 0.03 microns (median = 7.0) for oat cell cases and 9.5 +/- 0.06 microns (median = 9.0) for intermediate cell cases (P less than .001). In 28.6% of these patients, the nuclear diameter overlapped in the range of 8 microns to 9 microns between both subtypes. Comparisons between the nuclear diameter of primary and metastatic SCLC cells revealed no statistically significant differences. The nuclear diameter of malignant cells correlated with the mitotic index and stage of disease, but did not correlate with the other nuclear morphologic variables or with survival. The only identified prognostic factor was the stage of disease; these results indicate that the nuclear diameter of malignant cells should not be considered a prognosticator or a guide for therapy in SCLC patients.     

Increased thermoresistance developed during growth of small multicellular spheroids

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (approximately 70 micron diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45 degrees C for periods less than or equal to 2 hr and at 42.5 degrees C for less than or equal to 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of 1) the extent of intercellular contact in spheroids of different cellular multiplicities, 2) differences in membrane damage influenced by trypsin heat treatment sequence, and 3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.     

Radiation-induced division delay in 9L spheroid versus monolayer cells

The technique of percentage labeled mitoses was used to compare radiation-induced division delay in 9L rat gliosarcoma cells growing as spheroids or as exponential monolayers. The length of delay induced by each of five X-ray doses was determined as the difference between control and irradiated cultures in the time required to reach the half-height of the first peak of labeled mitoses. Spheroid cells were delayed significantly longer than monolayer cells; the slopes of the dose responses were 32 and 13 min/Gy, respectively. Cells in small spheroids (150 micron diameter) were delayed to the same extent as cells in large spheroids (800 micron diameter). Like the contact effect previously observed as enhanced radiation survival of cells grown as spheroids, the increased radiation-induced delay may be a consequence of the growth of cells in three-dimensional contact.     

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.     

The role of low intracellular or extracellular pH in sensitization to hyperthermic radiosensitization

Previous work showed that intracellular pH (pHi) and not extracellular pH (pHe) was the determinant in the low pH sensitization of hyperthermic killing. The present studies show that the same is true for heat-induced radiosensitization and loss of cellular DNA polymerase activities. Chinese hamster ovary cells after they had adapted to low pH (6.7) had an increase in pHi which rendered cells partially resistant to the low pH sensitization of heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities. These results were quantified by plotting versus pHe, both the thermal enhancement ratio (TER), defined as the ratio of the X-ray dose without heat to the X-ray dose with heat to give an isosurvival value of 0.01, and the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to the D0 of the radiation survival curve for heat plus radiation. Both the TER and TEF were higher for the unadapted cells than for the adapted cells, i.e., 1.3-1.4 fold higher at a pHe of 6.3. However, the TER or TEF plotted versus pHi was identical for the two cell types. Finally, heat-induced loss of cellular DNA polymerase activities correlated with pHi and not pHe. Therefore, we conclude that pHi and not pHe is responsible for the increase by acid in heat-induced radio-sensitization and loss of cellular DNA polymerase activities.     

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.     

Integration of hyper-compliant microparticles into a 3D melanoma tumor model

Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15–30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases.     

Acrylamide sensitization of the heat response of the cytoskeleton and cytotoxicity in attaching and well-spread synchronous Chinese hamster ovary cells

The vimentin intermediate filament (VIMF) network is more sensitive to heat-induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1 Chinese hamster ovary (CHO) cells (Coss and Wachsberger: Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert: European Journal of Cell Biology 37:169-174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1 or S phase) or in the well-spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival. CHO cells, either in the process of spreading or in the well-spread state, were sensitized to heat-induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1 phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1 but not necessarily to the death of cells treated in S phase. The degree of acrylamide sensitization of heat-induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well-spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment and spreading.     

Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake.

31P- and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4 +/- 1 fmol/cell compared to 8 +/- 1 fmol/cell in small (150 microns) proliferating spheroids (P less than 0.0002). The average PC pool size at steady state was reduced to 11 +/- 6 fmol/cell compared to 22 +/- 8 (P less than 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) (P less than 0.0001) for proliferating cells. The rate constant of CTP:phosphocholine cytidyltransferase (0.05 h-1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2-0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 microM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P less than 0.07). The rate constant of CTP:phosphoethanolamine cytidyltransferase (0.07 h-1) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2-0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.     

收缩活动促进新生大鼠培养心室肌细胞的^3H—亮氨酸...

To determine whether contraction could influence cell growth, the rate of protein synthesis (3H-leucine incorporation) and cell diameter and volume were measured in cultured neonatal rat cardiac myocytes beating spontaneously or arrested by high potassium. In medium supplemented with 10% calf serum, the 3H-leucine incorporation for 24 h in contracting myocytes (CMC) was significantly higher by 14.2% than that in quiescent myocytes (QMC), i.e. 1,229 +/- 29 cpm/10(5) cells vs. 1,076 +/- 60 cpm/10(5) cells (P < 0.01, n = 5 for each group). The cell diameter and cell volume in QMC group were respectively 15.14 +/- 0.42 microns and 1,842 +/- 123 microns3, while in the CMC group the corresponding figures reached to 16.82 +/- 0.64 microns3 and 2,495 +/- 210 microns3, increased by 11.1% and 35.5% respectively (P < 0.01, n = 6 for each group). With prolongation of culture time, the differences in these parameters between CMC and QMC became even more significant. In all these experiments, there was no significant difference in cell number between the two groups (P > 0.05). It is concluded that contraction per se can accelerate protein synthesis and cell growth in neonatal rat ventricular myocardium.     

Decay of thermal resistance following acute heating is independent of the G1- to S-phase transition

The phenomena of heat-induced G1 delay and thermal resistance were compared in synchronous populations of CHO cells. Mildly toxic induction doses of 5 min (Single cell survival, (SCS) = 0.90 +/- 0.06) and 10 min (SCS = 0.69 +/- 0.12) at 45 degrees C resulted in G1 delays of 4.3 and 11.3 h, respectively. Thermal resistance was tested (30 min, 45 degrees C) for up to 32-92 h following the induction dose. Thermal resistance did not start to decay prior to 26 h following the induction dose. These data confirm reports by R. R. Read, M. H. Fox, and J. S. Bedford [Radiat. Res. 98, 491-505 (1984)] and G. L. Rice, J. W. Gray, P. N. Dean, and W. C. Dewey [Cancer Res. 44, 2368-2376 (1984)] that acutely heated G1 populations of CHO cells progress into S phase without a concurrent loss of thermal resistance, using 45 degrees C induction doses even less toxic than used by other workers.     

Relationship between heat sensitivity and polyamine levels after treatment with alpha-difluoromethylornithine (DFMO)

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to study the effect of polyamine depletion on delayed heat sensitization in Chinese hamster ovary cells (CHO). The cells were treated with 1 or 10 mM DFMO for 8 or 48 h and then given a single heat treatment (43 degrees C, 90 min) at intervals up to 150 h after DFMO addition. Cellular survival, DNA polymerase activity, and polyamine levels were measured. Delayed heat sensitization for cell lethality began 50-55 h (about two cell divisions) after addition of 10 or 1 mM of DFMO for 8 or 48 h, respectively; i.e., cell survival of heated control cells was about 10(-1), but decreased to 10(-4)-10(-5) in heated DFMO-treated cells by 100 h. During this same interval, delayed heat sensitization also was observed for loss of DNA polymerase beta activity (from 20% in cells heated without DFMO treatment to 7% in heated DFMO-treated cells), but none was observed for DNA polymerase alpha activity. Delayed heat sensitization disappeared at 120-130 h after DFMO addition, with survival of heated DFMO-treated cells returning to that for heated control cells. The onset of delayed heat sensitization occurred 30-40 h after intracellular levels of putrescine and spermidine were depleted by more than 95%; however, spermine levels were not lowered, and in some cases even increased. Levels of putrescine and spermidine increased 5-10 h before delayed heat sensitization disappeared. While putrescine reached 25% of control, spermidine exceeded control levels during this time. Furthermore, delayed heat sensitization could be reversed by adding 10(-3) M putrescine or 5 X 10(-5) M spermidine 85-95 h after DFMO addition; in both cases spermidine increased 5-10 h before the decrease in heat sensitization. Finally, neither delayed heat sensitization nor depletion of spermidine was observed in nondividing plateau-phase cells treated with DFMO, although putrescine was depleted. These results lead to the hypothesis that DFMO-induced heat sensitization which occurs after inhibition of the synthesis of putrescine is secondary to the depletion of spermidine in some critical compartment of the cell or to a biochemical alteration. This depletion or biochemical alteration apparently occurs as the cells divide about two times after the intracellular levels of soluble spermidine have been depleted.