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Background Improvement of radiotherapy efficacy requires better insight in the dynamic responses that occur during irradiation. Here, we aimed to identify the molecular responses that are triggered during clinically applied fractionated irradiation. Methods Gene expression analysis was performed by RNAseq or microarray analysis of cancer cells or xenograft tumors, respectively, subjected to 3–5 weeks of 5 × 2 Gy/week. Validation of altered gene expression was performed by qPCR and/or ELISA in multiple cancer cell lines as well as in pre- and on-treatment biopsies from esophageal cancer patients (NCT02072720). Targeted protein inhibition and CRISPR/Cas-induced gene knockout was used to analyze the role of type I interferons and cGAS/STING signaling pathway in the molecular and cellular response to fractionated irradiation. Results Gene expression analysis identified type I interferon signaling as the most significantly enriched biological process induced during fractionated irradiation. The commonality of this response was confirmed in all irradiated cell lines, the xenograft tumors and in biopsies from esophageal cancer patients. Time-course analyses demonstrated a peak in interferon-stimulated gene (ISG) expression within 2–3 weeks of treatment. The response was accompanied by a variable induction of predominantly interferon-beta and/or -lambda, but blocking these interferons did not affect ISG expression induction. The same was true for targeted inhibition of the upstream regulatory STING protein while knockout of STING expression only delayed the ISG expression induction. Conclusions Collectively, the presented data show that clinically applied fractionated low-dose irradiation can induce a delayed type I interferon response that occurs independently of interferon expression or STING signaling. These findings have implications for current efforts that aim to target the type I interferon response for cancer treatment.

Background and purpose: Effective combination treatments with fractionated radiotherapy rely on a proper understanding of the dynamic responses that occur during treatment. We explored the effect of clinical fractionated radiotherapy on the development and timing of radioresistance in tumor cells. Methods and materials: Different colon (HT29/HCT116/COLO320/SW480/RKO) and high-grade astrocytoma (D384/U-251MG) cancer cell lines were treated for 6 weeks with daily fractions of 2 Gy, 5 days per week. Clonogenic survival was determined throughout the treatment period. In addition, the radiosensitivity of irradiated and non-irradiated was compared. Finally, the effect of different dose fractions on the development of radioresistance was determined. Results: All cell lines developed radioresistance within 2–3 weeks during fractionated radiotherapy. This was characterized by the occurrence of a steady state phase of clonogenic survival. In U-251MG cells this was accompanied by increased cell senescence and stemness. After recovering from six weeks of treatment, the radiosensitivity of fractionally irradiated and non-irradiated cells was similar. Including transient radioresistance, described as (α/β)−( d +1), as a factor in the classic LQ model resulted in a perfect fit with the experimental data observed during fractionated radiotherapy. This was confirmed when different dose fractions were applied. Conclusions: Fractionated irradiation of cancer cells in vitro following clinical radiation schedules induces a reversible radioresistance response. This adaptive response can be included in the LQ model as a function of the dose fraction and the alpha/beta-ratio of a given cell line. These findings warrant further investigation of the mechanisms and clinical relevance of adaptive radioresistance.

Purpose There is progressing evidence for the anti-cancer potential of the natural compound and dietary spice curcumin. Curcumin has been ascribed to be cytotoxic for various tumour cell types, to inhibit cell proliferation and to interfere with the cellular oxidant status. The compound has been notified as a therapeutic agent with radiosensitizing potential in brain tumour therapy. We considered the rationale to combine curcumin with radiation in the treatment of human glioblastoma multiforme (GBM). Method Determination of clonogenic cell survival following exposure of U251 human glioma cells to single dose (1–6 Gy) and fractionated irradiation (5 daily fractions of 2 Gy) without and with curcumin. Additional literature search focused on the interaction between curcumin and radiotherapy in experimental and clinical studies on human glioma. Results No interaction was found on the survival of U251 human glioma cells after irradiation in combination with curcumin at clinically achievable concentrations. Experimental in vitro and in vivo data together with clinical bioavailability data from the literature do not give evidence for a radiosensitizing effect of curcumin. Reported GBM intratumoural curcumin concentrations are too low to either exert an own cytotoxic effect or to synergistically interact with radiation. Novel approaches are being explored to increase the bioavailability of curcumin and to facilitate transport over the blood–brain barrier, aimed to reach therapeutic curcumin levels at the tumour site. Conclusion There is neither a biological nor clinical rationale for using curcumin as radiosensitizer in the therapy of GBM patients.

The extent of tumor oxygenation is an important factor contributing to the efficacy of radiation therapy (RTx). Interestingly, several preclinical studies have shown benefit of combining RTx with drugs that inhibit tumor blood vessel growth, i.e. angiostatic therapy. Recent findings show that proper scheduling of both treatment modalities allows dose reduction of angiostatic drugs without affecting therapeutic efficacy. We found that whilst low dose sunitinib (20 mg/kg/day) did not affect the growth of xenograft HT29 colon carcinoma tumors in nude mice, the combination with either single dose RTx (1x 5Gy) or fractionated RTx (5x 2Gy/week, up to 3 weeks) substantially hampered tumor growth compared to either RTx treatment alone. To better understand the interaction between RTx and low dose angiostatic therapy, we explored the effects of RTx on tumor angiogenesis and tissue perfusion. DCE-MRI analyses revealed that fractionated RTx resulted in enhanced perfusion after two weeks of treatment. This mainly occurred in the center of the tumor and was accompanied by increased tissue viability and decreased hypoxia. These effects were accompanied by increased expression of the pro-angiogenic growth factors VEGF and PlGF. DCE-MRI and contrast enhanced ultrasonography showed that the increase in perfusion and tissue viability was counteracted by low-dose sunitinib. Overall, these data give insight in the dynamics of tumor perfusion during conventional 2 Gy fractionated RTx and provide a rationale to combine low dose angiostatic drugs with RTx both in the palliative as well as in the curative setting.

Introduction To improve radiotherapy efficacy, insight in the dynamic biological responses that occur during clinical radiation schedules is vital. In the current study we explored gene expression changes during the course of fractionated radiotherapy (FrRT) that might be targeted with combination treatment strategies. Methods Human cell lines HT29, SW480, HCT116, COLO320, RKO (colorectal carcinoma), D384 (glioblastoma) and OE19 (esophageal adenocarcinoma) as well as HT29 xenograft tumors in nude mice were irradiated up to 3 to 5 weeks with daily fractions of 2 Gy for 5 days per week. In vivo tumor growth was monitored daily and in vitro clonogenic survival was determined every other day. At the end of each treatment week tumors and cells were harvested for RNA isolation. Subsequently, gene expression analysis was performed by RNA sequencing and micro-array analysis. Altered gene expression levels were validated by qPCR. Protein expression was determined by ELISA and Western blot. Results Tumor growth of the HT29 xenografts declined after 2 weeks of FrRT compared to non-irradiated tumors. In irradiated cell lines, clonogenic survival analyses revealed a log-linear decline in survival in the first 2 weeks, after which a steady-state-like phase was reached up to 5 treatment weeks. Gene expression analysis of both xenograft tumors as well as irradiated cell lines at this 2 week time point identified a type-1 interferon (IFN) mediated signaling pathway as the most significantly enriched biological process. The induction of a selection of IFN-stimulated genes (ISGs) was confirmed by qPCR. Comparable responses were observed in both STING positive (HT29, SW480, HCT116, D384) and negative (COLO320, RKO, OE19) cell lines. To get more insight in the dynamics of the IFN response we analyzed ISGs expression at the end of each treatment week. This revealed a peak in ISGs expression after 10 to 15 fractions, both in vivo and in vitro. The ISGs expression levels decreased with continuation of treatment but always remained above the basal level of non-irradiated cells. Single dose irradiation up to 10 Gy also induced dose-dependent ISGs expression, albeit typically 5 to 10 fold lower as compared to FrRT. Subsequent protein and mRNA expression analyses consistently revealed induction of IFN beta expression, with peak levels measured after 10 to 15 fractions. In addition, occasional induction of IFN lambda 2/3 protein and mRNA expression was observed. Conclusion FrRT induces an intrinsic type-1 IFN response which occurs independent of STING. This response is associated with induction of IFN beta expression, which peaks after 2 to 3 weeks of treatment and coincides with a shift in in vivo tumor growth and in vitro clonogenic survival. Targeting this response during FrRT might be exploited as a novel combination treatment. Citation Format: Ruben S. Goedegebuure, Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Sarah Derks, Henk M. Verheul, Ben J. Slotman, Adrian Harris, Victor L. Thijssen. A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3738.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6andnbsp;Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72andnbsp;h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24andnbsp;h for all cell lines. Exposure to 750andnbsp;μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non–small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle–related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2–M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. Mol Cancer Ther; 14(6); 1434–44. ©2015 AACR.

Investigation of clonogenic cell survival and cell proliferation following single dose and fractionated delivery of high dose rate flattening filter free (FFF) irradiation compared to conventional dose rates.The human astrocytoma D384, glioma T98 and lung carcinoma SW1573 cell lines were irradiated using either a single dose (0-12 Gy) or a fractionated protocol of 5 daily fractions of 2 Gy (D384) or 3 Gy (SW1573). Cells were irradiated inside a phantom using fixed gantry beams of a linear accelerator. A sliding window technique created homogeneous dose distributions over the surface of the cell cultures. Irradiations using standard beams (6 MV, 600 MU/min.) and high dose rate FFF beams (10 MV, 2400 MU/min.) were compared. Cell survival was determined by clonogenic assay. In the fractionated irradiation set-up, the number of clonogenic cells was estimated by including tumor cell proliferation during the overall treatment time in the analysis.All cell lines showed equal cell survival following irradiation using either the FFF beams or conventional flattened (FF) beams. This was observed after single dose exposure (0-12 Gy) as well as after fractionated irradiation (p = 0.08 for D384 and 0.20 for SW1373 cell lines).FFF irradiation with a dose rate of 2400 MU/min and four times higher dose per pulse compared to irradiation with FF beams did not change cell survival for three human cancer cell lines up to a fraction dose of 12 Gy compared to irradiation using FF beams.

PURPOSE: This study was performed to determine the effects of a continuous exposure to paclitaxel (taxol) in combination with fractionated irradiation on cell proliferation and survival.METHODS AND MATERIALS: Human lung carcinoma cells (SW1573) were given a daily treatment with 3 Gy of x-rays during 5 days in the continuous presence of 5 nM taxol. The surviving fraction and the total number of cells were determined every 24 h before and immediately after irradiation.RESULTS: Irradiation with 5 x 3 Gy and 5 nM taxol cause approximately the same inhibition of cell proliferation. In combination these treatments have an additional effect and the cell population increases no further after the first 24 h. Whereas the cells become more resistant to taxol after the first 24 h with a minimum survival of 42%, taxol progressively reduces the population of surviving cells in combination with x-rays when the number of fractions increases, up to 25-fold relative to irradiation alone. The enhancement effect of 5 nM taxol is likely to be attributed to an inhibition of the repopulation during fractionated irradiation and not to an increased radiosensitivity. Only after treatment with 10 or 100 nM taxol for 24 h, which is attended with a high cytotoxicity, is moderate radiosensitization observed.CONCLUSION: Taxol, continuously present at a low concentration with little cytotoxicity, causes a progressive reduction of the surviving cell population in combination with fractionated irradiation, mainly by an inhibition of the repopulation of surviving cells between the dose fractions.

Temozolomide (TMZ) is given in addition to radiotherapy in glioma patients, but its interaction with the commonly prescribed antiepileptic drug valproic acid (VPA) is largely unknown. Induction of DNA demethylation by VPA could potentially induce expression of the O-6-methylguanine-DNA-methyltransferase (MGMT) protein, causing resistance to TMZ and thereby antagonizing its effect. Therefore, this study investigates the interaction between VPA, TMZ, and gamma-radiation. Two glioma cell lines were used that differ in TMZ sensitivity caused by the absence (D384) or presence (T98) of the MGMT protein. VPA was administered before (24/48 h) or after (24 h) single doses of gamma-radiation; or, after 24 h, VPA treatment was accompanied by a single dose of TMZ for another 24 h. For trimodal treatment the combination of VPA and TMZ was followed by single doses of gamma-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF0.2 1.4 and 1.5) but not after postirradiation (DMF0.2 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF0.2 1.7) in both the TMZ-sensitive cell line (D384) and the TMZ-resistant cell line (T98). The combination of VPA and TMZ caused a significant radiation enhancement (DMF0.2 1.9 and 1.6) that was slightly more effective than that of VPA alone. VPA does not antagonize the cytotoxic effects of TMZ. Preincubation with VPA enhances the effect of both gamma-radiation and TMZ, in both a TMZ-sensitive and a TMZ-resistant human glioma cell line. VPA combined with TMZ may lead to further enhancement of the radiation response

PURPOSE: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated gamma-irradiation.METHODS AND MATERIALS: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0-6 Gy) and daily fractionated doses (2 Gy per fraction) of gamma-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell-survival curves were determined for both the single-dose and the fractionated irradiation experiments. To establish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells.RESULTS: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 micromol/L (AMC-3046), 3 micromol/L (VU-109), and 2.5 micromol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect.CONCLUSIONS: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to gamma-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene.

A common feature of Glioblastoma Multiforme (GBM) is an augmented RTK/RAS/PI3K pathway signaling. This pathway has been shown to be involved in DNA repair by both NHEJ and HR in various tumor types and is thought thereby to interfere with its chemo- and radioresistance. Targeting this pathway to sensitize GBM to chemo- and radiotherapy seems to be an attractive therapeutic approach. Proliferation and clonogenic survival of U251, U87 and T98 GBM cells were assessed following exposure to, BKM120 (pan class I PI3K inhibitor), RAD001 (mTORC1 inhibitor), MK-2206 (AKT inhibitor) and/or MEK162 (ERK inhibitor) alone and in combination with either irradiation or Temozolomide (TMZ). Effectivity of each compound on its target pathway was assessed by westernblot. BEZ235 (PI3K/mTOR/DNA-PK inhibitor) was used as a positive control for radiosensitization. Our data show BKM120 and MK-2206 to effectively inhibit AKT phosphorylation at 1 uM and leads to growth inhibition in all tested cell lines. However, these compounds are not toxic on clonogenic cell survival and do not sensitize the cells to TMZ or irradiation. Increasing the dose to 5 uM and 10 uM for BKM120 and MK-2206 respectively an antagonistic effect was found in the combination with TMZ and irradiation. Effectively inhibiting mTOR, the downstream effector of AKT, by 100 nM RAD001 also does not radiosensitize GBM cells. Inhibiting the parallel ERK-pathway by 1 uM MEK162, dephosphorylates ERK1/2 and leads to a slight increase in AKT phosphorylation. Inhibiting both pathways with BKM120 and MEK162 resulted in a synergistic growth inhibition of U251 cells however no radiosensitizing effect was found in a clonogenic assay. In conclusion, selective targeting of key components in the AKT- and/or ERK signaling pathway effectively inhibits proliferation of GBM cells yet does not sensitize them to either TMZ or radiotherapy, indicating that GBM does not depend on the RTK/RAS/PI3K pathway for its DNA-repair. Citation Format: Ravi Narayan, Rogier Dik, Jaap van den Berg, Lukas J.A. Stalpers, Brigitta G. Baumert, Ben J. Slotman, Peter Sminia. AKT- and ERK signaling inhibition does not sensitize GBM cells to chemo-/radiation in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4914. doi:10.1158/1538-7445.AM2014-4914

Purpose: To investigate the effect of temozolomide, a 3-methyl derivative of mitozolomide in combination with X-rays in human glioma-derived cell lines. Methods and Materials: Glioma cell lines D384 and U251 were treated with temozolomide for various periods of time in combinations with X-rays. Temozolomide administration was repeated every 24 h for exposures up to 96 h. Cytotoxicity was determined with a clonogenic assay. Results: Incubation of D384 cells with temozolomide during 24 h prior to or following irradiation results in a moderate enhancement of the cytotoxicity. Prolonged treatment with temozolomide, i.e., 48–96 h before X-rays, causes a stronger potentiation. In contrast, no enhancement is observed in irradiated U251 cells in combination with 24–96 h temozolomide treatment. In addition to single-dose irradiation, we investigated the effect of temozolomide in D384 cells with concomitant fractionated irradiation. A 96-h exposure to temozolomide with simultaneous doses of 2 Gy X-rays at 24-h intervals, causes a significant further reduction in cell survival as compared to fractionated irradiation only. Conclusion: Depending on the cell line, treatment of glioma cells with temozolomide and X-rays can have either an additional effect or potentiate cell killing.

Introduction: Over 50% of all cancer patients receive radiotherapy during their treatment. Recent studies demonstrate that combining radiotherapy with angiostatic drugs may have a beneficial effect on tumor growth inhibition in vivo. However, efficacy depends on the type of drug, the dose and the scheduling. We adapted the chick chorioallantoic membrane (CAM) assay to allow rapid analysis of the effects of radiotherapy in combination with angiostatic drugs on in vivo angiogenesis. Using this model, we studied the effects of different treatment schedules with sunitinib on tumors grafted onto the CAM. Methods: The CAMs of fertilized chicken eggs were treated daily with sunitinib (50 μL of 10 μM) starting at embryonic day of development (EDD) 6. Irradiation was given using a cobalt-60 source. On EDD10 the CAM vasculature was imaged and analyzed using specific software (HetCAM, DCI labs). For the in vivo tumor model, 5x106 HT29 or D384 cells were grafted on the CAM on EDD6. From EDD10 onwards, combination therapy was applied to the tumors using different treatment schedules. Tumor growth was measured daily until EDD17 after which tumor were dissected for further analysis. Results: Single dose (SD) radiotherapy (4 Gy) on EDD6 induces an almost 50% reduction in the vessel length and the number of branchpoints. Within 3 days these vascular parameters normalize. Sunitinib treatment for 2 and 4 days results in significant reduction of the vascular parameters. Applying 4 Gy after sunitinib treatment has an additive effect on the vasculature. In the tumor grafts, SD radiotherapy (4 Gy) decreased tumor volume and weight significantly four days after irradiation which was mainly caused by impaired tumor cell growth rather than by vascular defects. Combining radiotherapy (SD of 4Gy) with sunitinib treatment (4x 50 uL of 0.8uM) only had a beneficial effect when irradiation was given within the first 3 days of sunitinib treatment. Furthermore, while SD irradiation with 2 Gy alone did not affect tumor growth, a beneficial effect was observed when 2 Gy irradiation was given at the start of sunitinib treatment. Conclusion: The effect of combining radiotherapy with sunitinib on in vivo angiogenesis and tumor growth depends on the treatment schedule. Optimal scheduling allows a reduction in radiation dose. Preclinical analysis of optimal dose/scheduling is essential for the design of clinical trials and for translation of combination therapy to the clinic. Citation Format: Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Richard Honeywell, Arjan W. Griffioen, Ben J. Slotman, Henk M. Verheul, Victor L. Thijssen. Improving the radio-sensitizing effect of sunitinib by drug-specific scheduling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2013-1586

Purpose: To investigate the role of the cyclic adenosie 3′:5′-monophosphate (AMP) signal transduction pathway in heat-induced cell death and the development of thermotolerance. Methods and Materials: Reuber H35 rat hepatoma cells were heated after preincubation with various compounds known to modulate the cyclic AMP signal transduction pathway. Cell survival was determining by colony-forming ability. Results: Preincubation of H35 cells with forskolin, a stimulator of adenylate cyclase, in combination with IBMX (3-isobutyl-1-methylxanthine), an inhibitor of cyclic AMP phosphodiesterase, results in thermosensitization. Similar results are obtained with various cyclic AMP analogs. Maximum thermosensitization occurs with 0.5 mM dibutyryl cyclic AMP (DBcAMP) after a preincubation period of 5 h and heating in the presence of the drug. The same relative degree of thermosensitization is found with 8-Cl-cAMP, but a 10-fold lower concentration Thermosensitization by DBcAMP is prevented by H89, a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Without additional cyclic AMP-inducing factors, H89 induces thermoprotection. Node of the drug treatments are cytotoxic at 37°C. DBcAMP does not affect the development of heat-induced thermotolerance but it reduces its experession to an extent similarly found in the observed thermosensitization in nonthermotolerant cells. Conclusion: The results strongly indicate that the cyclic AMP signal transduction pathway is involved in the process of heat-induced cell death. DBcAMP reduces the expression of thermotolerance, but does not affect its induction.