Nitric oxide is known to be a multifunctional physiological substance. Recently, it was suggested that nitric oxide is involved in p53-dependent response to many kinds of stress, such as heat shock and changes in cellular metabolism. To verify this hypothesis, we examined the effect of nitric oxide produced endogenously by heat-shocked cells on nonstressed cells using a human glioblastoma cell line, A-172, and its mutant p53 (mp53) transfectant (A-172/mp53). The accumulation of inducible nitric oxide synthase was caused by heat treatment of the mtp53 cells but not of the wild-type p53 (wtp53) cells. The accumulation of heat shock protein 72 (hsp72) and p53 was observed in nontreated mtp53 cells cocultivated with heated mp53 cells, and the accumulation of these proteins was suppressed by the addition of a specific inducible nitric oxide synthase inhibitor, aminoguanidine, to the medium. Furthermore, the accumulation of these proteins was observed in the wtp53 cells after exposure to the conditioned medium by preculture of the heated mp53 cells, and the accumulation was completely blocked by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. In addition, the accumulation of hsp72 and p53 in the wtp53 cells was induced by the administration of an nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, to the medium. Finally, the thermosensitivity of the wtp53 cells was reduced in the conditioned medium by preculture of the heated mp53 cells as compared with conventional fresh growth medium. Our finding of the accumulation of hsp72 and p53 in nitric oxide-recipient cells cocultivated with heated nitric oxide-donor cells provides the first evidence for an intercellular signal transduction pathway via nitric oxide as intermediate without cell-to-cell interactions such as gap junctions.

NO3 is an important regulatory substance for the immune response, cytotoxicity, neurotransmission, and vasodilatation (1). NO is endogenously generated from l-arginine by NOS isoenzymes (2). The activities of the constitutive forms neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3) are dependent on elevated cellular calcium concentrations (3). It is well known that iNOS (or NOS2) is expressed in various species of mammalian cells after exposure to many kinds of inducers such as cytokines, bacterial lipopolysaccharide, heat shock, and hypoxia (4, 5, 6). It has been reported that iNOS can produce sustained high concentrations of NO after induction (3). High concentrations of NO and NO metabolites have been shown to cause DNA damage and have been shown to be mutagenic (7, 8, 9). On the other hand, it has been shown that high concentrations of NO can quench superoxide anion radicals and that the reaction product, peroxynitrite, can rearrange to nontoxic nitrate. NO can also scavenge oxidizing intermediates generated from peroxynitrite (10). Recently, it has also been reported that NO can induce hsps and wtp53 protein, which play important roles in adaptation of the organism to many kinds of stress (11, 12).

The inducible hsp72 is one of the stress proteins induced transcriptionally by various environmental stressors such as heat shock, cold shock (13), UV (14, 15), ionizing radiation (14, 16), and DNA-damaging agents (17). Hsps play important roles in cellular thermoresistance and the development of thermotolerance as an adaptive response after exposure to heat shock (18, 19). It has been found that many stress proteins, including hsp72, function as molecular chaperones for repair of heat-denatured proteins (20). The wtp53 accumulated in response to genotoxic and nongenotoxic stressors, such as DNA-damaging agents and heat, respectively, has been shown to be a key protein acting as a cell cycle checkpoint (21, 22). Thus, it belongs to the signaling pathway by which cells regulate the G1-S transition after either genotoxic or nongenotoxic insults; in this way, wtp53 functions as a transcriptional factor to control the cell cycle, especially in stressed cells. Recently, it has been considered that the major functional role of wtp53 is as an integrator of stress-response signals (23). The p53-dependent cellular events, such as WAF1 induction by heat shock, cold shock, or low pH, have been demonstrated using a pair of human glioblastoma cell lines, A-172 and T98G, bearing wtp53 and mp53 genes, respectively (24, 25, 26). We have established a transfected cell line, A-172/mp53, by transfecting A-172 cells with the mp53 gene (27). The genotype of A-172/mp53 cells differs from that of A-172 cells only in the p53 gene. We have confirmed that the wtp53-dependent cellular events induced after hyperthermia do not occur in the A-172/mp53 cells (28).

Recently, NO is suggested to be involved in p53-dependent response to many kinds of stress such as heat shock and changes in cellular metabolism. In the present study, to verify this hypothesis we examined: (a) the kinetics of the accumulation of iNOS after heat treatment; (b) the kinetics of the accumulation of hsp72 and p53 in nontreated cells cocultivated with the heated cells; and (c) the thermosensitivities of cells in the conditioned medium by preculture of the heated cells using a human glioblastoma cell line, A-172, and its mutant p53 transfectant (A-172/mp53).

Chemicals.

SNAP was purchased from Molecular Probes, Inc. (Eugene, OR). c-PTIO was purchased from Doujin Chemical Co. (Tokyo, Japan). Both chemicals were dissolved at 10 mm in PBS(−) and stored at −20°C until use. The protein assay kit was purchased from Nacalai Tesuque (Kyoto, Japan). Anti-iNOS monoclonal antibody (Clone 6), anti-hsp72 monoclonal antibody (C92F3A-5), anti-p53 monoclonal antibody (PAb1801 or DO-1), and horseradish peroxidase-conjugated anti-mouse IgG antibody were purchased from Transduction Laboratories (Lexington, MA), StressGen (Victoria, British Columbia, Canada), Oncogene Science, Inc. (Uniondale, NY), and Zymed Laboratories, Inc. (South San Francisco, CA), respectively. The BLAST: Blotting Amplification System was purchased from DuPont/NEN Research Products (Boston, MA). Giemsa solution was purchased from Merck & Co., Inc. (Rahway, NJ).

Cells.

A human glioblastoma cell line, A-172, was purchased from JCRB Cell Bank (Setagaya, Tokyo, Japan). The two human glioblastoma cell lines, A-172 and A-172/mp53, were cultured in DMEM containing 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 50 μg/ml kanamycin (DMEM-10). A-172 cells have the wild-type p53 gene (wtp53), whereas A-172/mp53 cells have a mutated p53 gene (mp53; Refs. 27 and 29). The doubling times of A-172 and A-172/mp53 cells were about 24 and 22 h, respectively. Plating efficiencies of both cell lines were about 50%.

Heat Treatment.

Twenty h before heat treatment, exponentially growing cells were seeded at ∼106 cells/dish in 9-cm dishes or ∼105 cells/flask in 25-cm2 flasks containing DMEM-10 without irradiated feeder cells. Cells were washed once with DMEM-10 and then immersed into a water bath (model EPS-47; Toyo Seisakusho Co., Tokyo, Japan) preset at 44°C ± 0.1°C. After treatment, cells were incubated at 37°C in a conventional humidified CO2 incubator.

Cocultivation of Cells on Slide Glasses with Cells in Dishes.

Twenty h before heat treatment, exponentially growing cells were seeded at ∼106 cells/dish in 9-cm dishes containing DMEM-10 without irradiated feeder cells. At the same time, cells were seeded on slide glasses at 2 × 105 cells/slide glass. Cells in dishes wrapped with parafilm were heated at 44°C for 15 min. After heat treatment, slide glasses containing the nonheated cells were transferred into dishes containing the heated cells and then incubated at 37°C for up to 10 h. At various times, cells were harvested from slide glasses and dishes, and total proteins were extracted and subjected to Western blotting for hsp72 and p53.

c-PTIO Treatment.

Twenty h before treatment, exponentially growing cells were seeded at ∼106 cells/flask in 75-cm2 flasks containing DMEM-10 without irradiated feeder cells. Cells were washed twice with DMEM-10. After addition of DMEM-10 containing c-PTIO (10 or 50 μm), cells in flasks were heated at 44°C ± 0.1°C for 15 min. Subsequently, cells were incubated at 37°C in a conventional humidified CO2 incubator. Then, the conditioned medium containing c-PTIO was recovered 10 h after incubation. Twenty h before treatment, exponentially growing cells were seeded at ∼105 cells/flask in 25-cm2 flasks containing DMEM-10 without irradiated feeder cells. Cells were washed twice with DMEM-10 and then exposed to the conditioned medium containing c-PTIO for 10 h at 37°C. After the treatment, cells were harvested and used for Western blot analysis.

SNAP Treatment.

Twenty h before treatment, exponentially growing cells were seeded at ∼105 cells/flask in 25-cm2 flasks containing DMEM-10 without irradiated feeder cells. Cells were washed twice with DMEM-10 and then treated with SNAP (1–10 μm) in DMEM-10 for 10 h at 37°C. After the treatment, cells were harvested and used for Western blot analysis.

Western Blot Analysis.

The cells were suspended in RIPA buffer and then treated by freezing and thawing three times. The protein contents in the supernatants obtained after centrifugation were quantified with a commercial protein assay kit. An aliquot of proteins was subjected to Western blotting analysis for iNOS, hsp72, and p53. After electrophoresis on 10% polyacrylamide gels containing 0.1% SDS and electrophoretic transfer to polyvinylidene difluoride membranes, proteins on the membrane were incubated with anti-iNOS, anti-hsp72, or anti-p53 monoclonal antibodies. For visualization of the bands, we used horseradish peroxidase-conjugated anti-mouse IgG antibody and the BLAST: Blotting Amplification System. The relative amounts of iNOS, hsp72, and p53 were calculated from the scanning profiles using a Macintosh (LC 475) computer with the public domain NIH Image program.

Measurement of Nitrite Concentration in Medium.

Nitrite concentration in medium was measured according to the method of Saltzman (30). Forty μl of medium were mixed well with 960 μl of the reagent containing 0.5% sulfanilic acid, 0.002% N-1-naphthylethylenediamine dihydrochloride, and 14% acetic acid. After standing at room temperature for 15 min, absorbance of samples at 550 nm was measured. The solution of sodium nitrite dissolved in DMEM was used as an authentic solution.

Survival Curves.

The surviving cell fractions after hyperthermia were determined as colony-forming units and corrected by the plating efficiency of the nontreated cells as a control. Two replicate flasks were used per experiment, and two or more independent experiments were repeated for each survival point. Colonies obtained were fixed with methanol and stained with 2% Giemsa solution. Visible colonies composed of >50 cells after 10 days were counted as having grown from surviving cells. Values are the means of at least two independent experiments; bars (SEs) are shown when their values exceeded those of the symbols. The T0 value represents the treatment period required to reduce the survival to 1/e with the exponentially regressing portion of the survival curves. The Tq (quasi-threshold treatment period) is defined as the treatment period at which the straight portion of the survival curve, extrapolated backward, cuts the treatment period axis drawn through a survival fraction of unity.

Statistical Analysis.

Significant levels were calculated using the unpaired Student’s t test. P < 0.05 was considered statistically significant.

After heating at 44°C for 15 min, A-172 or A-172/mp53 cells (2 × 105 cells/flasks) were incubated at 37°C for up to 24 h, cells were harvested at various times, and total proteins were extracted and subjected to Western blotting for iNOS. Accumulation of iNOS was observed in A-172/mp53 cells but not in A-172 cells (Fig. 1). In A-172/mp53 cells, the level of iNOS increased gradually after the heating and reached a level about 3-fold greater than the control level at 24 h, whereas the level of iNOS in A-172 cells hardly increased after the heating.

We analyzed the nitrite concentration in the medium in which the heated A-172 and A-172/mp53 cells were cultured (Fig. 2). In the medium in which A-172/mp53 cells were cultured, the nitrite concentration before heating was about 0.6 μm. Just after heating at 44°C for 15 min, the nitrite concentration decreased to 0.3 μm, which is similar to the level in the medium in which A-172 cells were cultured before the heating. Subsequently, the nitrite concentration increased to over 1.6 μm 12 h after heating, and this level was maintained at least up until 24 h (Fig. 2,B). In addition, the elevation of nitrite concentration in the medium of A-172/mp53 cells was completely suppressed by the addition of aminoguanidine (100 μm). On the other hand, in the medium of A-172 cells, the nitrite concentration decreased, and the addition of aminoguanidine did not affect the nitrite concentration after heating (Fig. 2,A). These results in Figs. 1 and 2 indicate that in A-172/mp53 cells, NO generated by the accumulated iNOS after heating causes an elevation of nitrite concentration in the medium. Our results confirmed a previous report that iNOS appeared to have produced presumably up to micromolar concentrations of NO (31).

To determine whether the cellular stress response was induced by the elevation of the nitrite concentration in the medium, we examined the accumulation of hsp72 and wtp53 in the nontreated A-172 cells that were cocultivated with A-172/mp53 heated at 44°C for 15 min, in the presence or absence of 100 μm aminoguanidine as iNOS inhibitor (see “Materials and Methods”). In the absence of aminoguanidine, both hsp72 and wtp53 were accumulated in the nontreated A-172 cells cocultivated with the heated A-172/mp53 cells (Fig. 3). However, this accumulation was completely abolished by the addition of aminoguanidine to medium. In the heated A-172/mp53 cells, a remarkable accumulation of hsp72 was observed in either the presence or absence of aminoguanidine, whereas the level of mp53 remained relatively stable. These results indicate that NO produced in the heated A-172/mp53 cells induces the accumulation of hsp72 and wtp53 in the cocultivated A-172 cells, suggesting that NO may be a mediator for an intercellular signal transduction pathway through the medium, without cell-to-cell contact, in the stress response.

To confirm that the accumulation of hsp72 and wtp53 was induced by NO, we examined the accumulation of these proteins in A-172 cells using an NO-specific scavenger and an NO-generating agent. The conditioned medium of A-172/mp53 cells was prepared by culturing the cells for 10 h after heating them at 44°C for 15 min in the presence or absence of 10 or 50 μm c-PTIO as NO scavenger. The levels of hsp72 and wtp53 in A-172 cells increased markedly 10 h after exposure to the conditioned medium prepared by culturing A-172/mp53 cells for 10 h after heating at 44°C for 15 min in the absence of c-PTIO (Fig. 4, Lane 3), whereas the levels of these proteins did not change 10 h after exposure to the conditioned medium prepared by culturing A-172/mp53 cells for 10 h without heating (Fig. 4, Lane 2). The accumulation of these proteins was diminished by the addition of c-PTIO to the conditioned medium of the heated A-172/mp53 cells in a dose-dependent manner (Fig. 4, Lanes 4 and 5). Furthermore, we examined whether NO can induce the accumulation of hsp72 and wtp53 in A-172 cells. The accumulation of hsp72 was observed 10 h after exposure to 1 μm SNAP, and the level of hsp72 was decreased at higher SNAP concentrations (Fig. 5). The accumulation of wtp53 was observed 10 h after exposure to 1 μm SNAP, reached a peak at 5 μm, and was sustained at an elevated level up to 10 μm. The results in Figs. 4 and 5 indicate that the large amount of NO produced by the elevated level of iNOS in the heated A-172/mp53 cells is released into the medium, acts as a mediator of the intercellular signal transduction, and induces the accumulation of hsp72 and wtp53 in nontreated A-172 cells.

In an attempt to elucidate the effects of NO on the cellular thermosensitivity, we examined the thermosensitivity of A-172 cells in various media at 44°C (Fig. 6 and Table 1). A-172 cells in the conditioned medium by preculture of A-172/mp53 cells for 10 h after heating at 44°C for 15 min (CM-Δ) were more thermoresistant than those in fresh growth medium (GM) or the conditioned medium by preculture of A-172/mp53 cells for 10 h without heating (CM; Fig. 6). In GM, the T0 of A-172 cells was 11.3 min. In CM, the thermosensitivity of A-172 cells scarcely changed; the T0 of A-172 cells was 13.8 min. However, in the CM-Δ, the T0 of A-172 cells was 20.0 min (Table 1). The thermal dose modifying ratios in T0 were 1.2 and 1.8 for CM and CM-Δ, respectively. The reduction of thermosensitivity in CM-Δ was similar to that of the thermotolerant A-172 cells prepared by preheating at 44°C for 15 min 10 h before challenge heating, which is represented by the dashed line in Fig. 6. The results in Fig. 6 and Table 1 indicate that NO released from the heated A-172/mp53 cells can induce thermoresistance in the nontreated A-172 cells through intercellular signal transduction.

In summary, we found that: (a) the accumulation of hsp72 and p53 was induced in NO-recipient cells by cocultivation with the NO-donor cells; (b) the accumulation of these proteins was also induced by exposure of cells to the conditioned medium by preculture of the NO-donor cells; (c) the accumulation of these proteins was completely blocked by the addition of NO scavenger to the conditioned medium; and (d) the cellular thermosensitivity was reduced in the conditioned medium by preculture of NO-donor cells as compared with fresh growth medium. Our findings demonstrate that NO released from the donor cells can induce hsp72 and p53 accumulation in the cocultivated NO-recipient cells through intercellular signal transduction without cell-to-cell interactions such as gap junctions. Previously, we reported that the cellular content of wtp53 increased after heating in human glioblastoma cells having wtp53 gene (29). Therefore, the fact that the accumulation of iNOS was observed only in mp53 cells after heating suggests that wtp53 accumulated after heating may suppress iNOS synthesis. These results confirm previous studies that wtp53 transrepressed iNOS expression through inhibition of the iNOS promoter in a variety of cells in vitro(12) and in vivo(32). Malyshev et al.(33) reported that NO generated from a chemical agent induces hsp72 accumulation. Forrester et al.(12) have also reported that NO generated from a chemical agent induces wtp53 accumulation. It is well known that a half-life of NO is extremely short (about 6 s), and NO can react immediately with oxygen or reactive oxygen intermediates (34). Therefore, our findings suggest at least three possible pathways of the intercellular signal transductions after hyperthermia, which can be initiated by NO: (a) reaction products of NO, which released from the heated A-172/mp53 cells, may induce directly accumulation of hsp72 and p53 in nonstressed A-172 cells; (b) NO itself and/or reaction products of NO may activate GMP cyclase; subsequently, cGMP may induce the accumulation of hsp72 and wtp53 in nonstressed A-172 cells; and (c) NO itself and/or reaction products of NO may induce secretion of certain cytokines and/or growth factors in the heated A-172/mp53 cells; subsequently they may induce accumulation of hsp72 and p53 in nonstressed A-172 cells. Thus, we demonstrated the intercellular signal transduction initiated by NO production in the donor cells to effect responses in the recipient cells. However, it is still unknown what mediates the signal transduction. In addition, the intercellular signal transduction initiated and mediated by NO and its reaction products may link the intracellular signal transduction induced by not only extracellular stress such as heat shock and UV rays but also intracellular stress such as production of oxidants and changes in metabolism (35).

It is well known that hsps, including hsp72, contribute to cellular thermoresistance and thermotolerance development through their functions as molecular chaperones, in which they play an important role in the protein repair process for certain heat-denatured cellular proteins (20). Likewise, wtp53 plays an important role in safeguarding the genomic integrity of mammalian cells in response to cellular injury (23). Cellular injury can trigger an accumulation of wtp53, resulting in p53-mediated increases in expression of growth-regulatory genes and G1 growth arrest (21). Previously, we reported that wtp53 induces G1 arrest after heating (29). During the G1 arrest induced by wtp53 after heating, the induced and accumulated hsps may repair heat-denatured proteins, thereby enhancing survival of cellular injury induced by heat and may block heat-induced apoptosis (36). Recently, Gansauge et al.(37) reported that endogenous production of NO revealed a G1 arrest in all of the tested cells using human pancreatic carcinoma cell lines. Thus, the thermoresistance of the NO-recipient cells is brought about by hsp72 and wtp53 accumulation induced by NO from the donor cells through the intercellular signal transduction pathway.

Previously, to maintain a high plating efficiency of cells, a γ-irradiated or antibiotic-treated cell feeder layer was often used in colony formation assays for measurement of cellular sensitivity to radiation, chemicals, or hyperthermia (38, 39). Recently, however, it has been reported that the cell survival and growth of the target cells are greatly affected by the presence of a feeder layer of irradiated cells, and that the feeder layer suppresses apoptosis induced in the target cells by certain treatments (40, 41). Our results indicate a possible mechanism of the feeder layer effects and suggest that the experimental results obtained using a feeder layer may not represent the intrinsic cellular sensitivity to radiation, chemicals, or hyperthermia. Furthermore, we consider that our results may indicate a possible mechanism of the bystander effects in cancer gene therapy. When a fraction of the cancer cells in a tumor was transfected with wtp53 or the TNF α gene, overall growth suppression, angiogenesis suppression, and stimulation of apoptosis were observed in the tumor (42, 43, 44). NO induces apoptosis in a p53-dependent manner in many types of cells (45, 46). Therefore, an intercellular signal transduction pathway mediated by NO, without cell-to-cell interactions, may be involved in the mechanisms of bystander effects in cancer gene therapy. Finally, our results suggest that elucidation of the effects of the microenvironmental conditions in tumors on cellular responses after treatments is very important in the fundamental research concerning various cancer therapies, including gene therapy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas No. 09255217, Exploratory Research No. 09877170, Scientific Research (A) No. 09307015, and Scientific Research (C) No. 10670840 from the Ministry of Education, Science, Sports and Culture, Japan.

            
3

The abbreviations used are: NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; hsp, heat shock protein; wtp53, wild-type p53; mp53, mutant p53; SNAP, S-nitroso-N-acetylpenicillamine; c-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide.

Fig. 1.

Accumulation of iNOS in A-172 (•) and A-172/mp53 (▴) cells after heating. The cells were heated at 44°C for 15 min and then incubated at 37°C for 0–24 h. The level of iNOS in each sample was analyzed by Western blotting as described in “Materials and Methods.” The level in nontreated cells was used as a control. Bars, SE.

Fig. 1.

Accumulation of iNOS in A-172 (•) and A-172/mp53 (▴) cells after heating. The cells were heated at 44°C for 15 min and then incubated at 37°C for 0–24 h. The level of iNOS in each sample was analyzed by Western blotting as described in “Materials and Methods.” The level in nontreated cells was used as a control. Bars, SE.

Close modal
Fig. 2.

Change of nitrite concentration in medium of A-172 (A) or A-172/mp53 (B) cells. Nitrite concentration in medium was measured according to the method of Saltzman (30). ○ and ▵, in the absence of aminoguanidine; • and ▴, in the presence of aminoguanidine (100 μm).

Fig. 2.

Change of nitrite concentration in medium of A-172 (A) or A-172/mp53 (B) cells. Nitrite concentration in medium was measured according to the method of Saltzman (30). ○ and ▵, in the absence of aminoguanidine; • and ▴, in the presence of aminoguanidine (100 μm).

Close modal
Fig. 3.

Western blot analysis of hsp72 and p53 in A-172 cells cocultivated with heated A-172/mp53 cells. A-172/mp53 cells were heated at 44°C for 15 min. After transfer of A-172 cells on slide glasses to dishes of A-172/mp53 cells, cell cultures were incubated at 37°C for 1 to 10 h. AG, aminoguanidine.

Fig. 3.

Western blot analysis of hsp72 and p53 in A-172 cells cocultivated with heated A-172/mp53 cells. A-172/mp53 cells were heated at 44°C for 15 min. After transfer of A-172 cells on slide glasses to dishes of A-172/mp53 cells, cell cultures were incubated at 37°C for 1 to 10 h. AG, aminoguanidine.

Close modal
Fig. 4.

Accumulation of hsp72 and p53 in A-172 cells exposed to the conditioned medium of A-172/mp53 cells. A-172 cells were incubated at 37°C for 10 h in the conditioned medium of A-172/mp53 cells. Lane 1, in control growth medium; Lane 2, exposed to the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h; Lane 3, exposed to the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h after heating at 44°C for 15 min; Lane 4, as Lane 3 but in the presence of 10 μm c-PTIO; Lane 5, as Lane 4 but in the presence of 50 μm c-PTIO.

Fig. 4.

Accumulation of hsp72 and p53 in A-172 cells exposed to the conditioned medium of A-172/mp53 cells. A-172 cells were incubated at 37°C for 10 h in the conditioned medium of A-172/mp53 cells. Lane 1, in control growth medium; Lane 2, exposed to the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h; Lane 3, exposed to the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h after heating at 44°C for 15 min; Lane 4, as Lane 3 but in the presence of 10 μm c-PTIO; Lane 5, as Lane 4 but in the presence of 50 μm c-PTIO.

Close modal
Fig. 5.

Accumulation of hsp72 and p53 in A-172 cells exposed to SNAP. A-172 cells were incubated at 37°C for 10 h in the absence (Lane 1) and in the presence of 1 μm (Lane 2), 5 μm (Lane 3), or 10 μm (Lane 4) SNAP, followed by Western blot analysis.

Fig. 5.

Accumulation of hsp72 and p53 in A-172 cells exposed to SNAP. A-172 cells were incubated at 37°C for 10 h in the absence (Lane 1) and in the presence of 1 μm (Lane 2), 5 μm (Lane 3), or 10 μm (Lane 4) SNAP, followed by Western blot analysis.

Close modal
Fig. 6.

Thermosensitivity of A-172 cells in the conditioned media of A-172/mp53 cells. The thermosensitivity of A-172 cells was analyzed in control growth medium (○), in the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h (▵), and in the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h after heating at 44°C for 15 min (•) by a colony formation assay. ----, thermosensitivity of thermotolerant A-172 cells prepared by preculture at 37°C for 10 h after heating at 44°C for 15 min.

Fig. 6.

Thermosensitivity of A-172 cells in the conditioned media of A-172/mp53 cells. The thermosensitivity of A-172 cells was analyzed in control growth medium (○), in the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h (▵), and in the conditioned medium by preculture of A-172/mp53 cells at 37°C for 10 h after heating at 44°C for 15 min (•) by a colony formation assay. ----, thermosensitivity of thermotolerant A-172 cells prepared by preculture at 37°C for 10 h after heating at 44°C for 15 min.

Close modal
Table 1

Thermosensitivities of A-172 cells in various media

MediumT0 (min)Tq (min)Thermal dose modifyingratio in T0
Fresh growth medium 11.3 12.3 1.00 
CMa 13.8 13.0 1.22 
CM-Δb 20.0 13.6 1.77 
MediumT0 (min)Tq (min)Thermal dose modifyingratio in T0
Fresh growth medium 11.3 12.3 1.00 
CMa 13.8 13.0 1.22 
CM-Δb 20.0 13.6 1.77 
a

The conditioned medium prepared by culturing of A-172/mp53 cells for 10 h without heating.

b

The conditioned medium prepared by culturing of A-172/mp53 cells for 10 h after heating at 44°C for 15 min.

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