Tetsuro Tamamoto

p53-dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET

Purpose : To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. Materials and methods : Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/m p53 cells were established by transfection with mutated p53 (m p53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/ neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV w m -1) followed by heating at 44°;C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. Results : It was found that (1) there was no significant difference in cellular sensitivity between SAS/ neo and SAS/m p53 cells to LET radiation of >30 KeV w m -1, although the radiosensitivity of SAS/ neo cells to X-rays was higher (1.2-fold) than that of SAS/m p53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV w m -1 in SAS/ neo cells, although only additive thermal enhancement was observed in SAS/m p53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/ neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/ neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/m p53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. Conclusions : The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.

Restoration by glycerol of p53-dependent apoptosis in cells bearing the mutant p53 gene

PURPOSE Effective heat-induced cell death in cultured cells bearing a mutant p53 (mp53) gene was sought by glycerol treatment which led to conformational change from mp53 to wild-type p53 (wtp53) in p53-null murine fibroblasts transfected with mp53. MATERIALS AND METHODS Heat sensitivity was measured using a colony-forming assay. For heat-induced apoptosis, gel electrophoresis was applied to detect DNA fragmentation and Hoechst33342 staining for apoptotic bodies. Glycerol (0.6 M) was applied to the cultured cells 48 h before heating at 44 degrees C in a water bath. RESULTS Wtp53 transfectants (MT158/wtp53-1) were sensitive to heat stress compared with mp53 transfectants (MT158/mp53-2 cells), and the combined treatment with glycerol enhanced cell killing only in the MT158/mp53-2 cells. After glycerol pretreatment for 48 h, the subsequent heat treatment enhanced DNA fragmentation and apoptosis in MT158/mp53-2 cells, while DNA fragmentation and apoptotic bodies were not enhanced with heat treatment alone in these cells. In contrast, DNA fragmentation or apoptotic bodies were clearly observed in MT158/wtp53-1 cells 3-24h after heat treatment. Treatment with glycerol alone did not induce apoptosis in the transfectants. CONCLUSIONS Glycerol appears to function as a chemical chaperone that restores mp53 to wtp53 function.

The dependence of p53 on the radiation enhancement of thermosensitivity at different let

PURPOSE The aim of this study is to investigate the dependence of p53-gene status on the radiation enhancement of thermosensitivity at different levels of linear energy transfer (LET). METHODS AND MATERIALS We used two kinds of human glioblastoma transfectants of A-172 cells bearing the wild-type p53 gene, A-172/neo cells with control vector containing the neo gene and A-172/mp53 cells with both the dominant negative mutated p53 gene and neo gene. We exposed these cells to X-rays and accelerated carbon-ion (C-) beams (13-200 KeV/microm) followed by heating at 44 degrees C. Cellular sensitivities were determined using clonogenic assay. RESULTS The radiation enhancement of thermosensitivity was LET-dependent for the A-172/neo cells, but this was not clearly demonstrated in the A-172/mp53 cells. The supraadditive radiation enhancement of thermosensitivity was observed in A-172/neo cells at the LET range of 13 to 70 KeV/microm, though only an additive effect was observed at higher LET. In A-172/mp53 cells, only an additive effect was observed through all the LET examined. CONCLUSION These results indicate that the radiation enhancement of thermosensitivity is p53- and LET-dependent. Our results suggest that the combined use of high-LET radiation and hyperthermia brings useful application for cancer therapeutic purposes.

WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

Purpose : There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as gamma-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53 -dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. Materials and methods : A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wt p53) and T98G bearing mutated p53 (m p53) . A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a m p53 expression vector respectively. Results : The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wt p53 cells (A-172 and A-172/neo) were more sensitive to X-rays than m p53 cells (A-172/mp53 and T98G). Conclusions : These results indicate that C-beams and alpha-particles induce p53 -dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.PURPOSE There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as y-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53-dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. MATERIALS AND METHODS A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wtp53) and T98G bearing mutated p53 (mp53). A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a mp53 expression vector respectively. RESULTS The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0 Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A-172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wtp53 cells (A-172 and A-172/neo) were more sensitive to X-rays than mp53 cells (A-172/mp53 and T98G). CONCLUSIONS These results indicate that C-beams and alpha-particles induce p53-dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.

p53- dependent hyperthermic enhancement of tumour growth inhibition by X-ray or carbon-ion beam irradiation

To elucidate p53 -dependency on combined treatment with radiation and hyperthermia, growth inhibition and apoptosis were analysed using transplantable human tumour. Human head and neck squamous cell carcinoma (HNSCC) cells carrying different p53 genes were transplanted into the thigh of nude mice. When the mean diameter of tumour reached 5-6 mm, the tumours were exposed to X-rays (2 Gy) or Carbon-ion (C-) beams (1 Gy) followed by heating at 42°C for 20 min. Tumour growth inhibition was evaluated by measuring the diameters of tumour. The induction of apoptosis and accumulation of apoptosis-related proteins were also analysed by immunohistochemical staining. Synergistic enhancement of tumour growth inhibition by hyperthermia was observed in wild-type p53 tumours treated with X-rays or C-beams but not in mutant p53 tumours. The incidence of apoptotic cells and activated-caspase-3-positive cells after combined treatment with them were significantly high in wild-type p53 tumours compared with that in mutant p53 tumours. The hyperthermic enhancement of tumour growth inhibition by X-ray- or C-beam-irradiation was p53 -dependent, suggesting that it might be highly correlated with p53 -dependent apoptosis.

Prognostic Significance of Muscle Attenuation in Pancreatic Cancer Patients Treated with Neoadjuvant Chemoradiotherapy

BackgroundEmerging evidences have gradually revealed the skeletal muscle attenuation (MA) was not only reflected the accumulation of lipids in skeletal muscle but also associated with physiological and pathological states. The aim of this study was to evaluate the impact of MA on the prognosis of pancreatic cancer patients treated with neoadjuvant chemoradiotherapy (NACRT).MethodsEighty-three patients with pancreatic cancer who received NACRT were enrolled. Patients were divided according to their Hounsfield units of the skeletal muscle at the third lumbar vertebra in CT. The lower quartile was defined as MA group and the remainder as control group.ResultsThere was no significant difference in overall survival between pre-NACRT MA and control groups. In contrast, patients with post-NACRT MA had a significantly poorer prognosis than patients without. The patients in the post-NACRT MA group were significantly older than patients in the control group. There were no significant differences in most clinicopathological and perioperative factors between both groups. However, patients with post-NACRT MA had a longer hospital stay than patients without. Furthermore, the incompletion rate of the proposed adjuvant chemotherapy was significantly higher in the MA group than control. Importantly, multivariate analysis indicated that post-NACRT MA was an independent prognostic factor.ConclusionsMuscle attenuation may have a significant impact in pancreatic cancer patients treated with multimodal therapy. Therefore, our data may provide new insights into perioperative patient care to improve the prognosis of resectable pancreatic cancer.

Optimal indication of neoadjuvant chemoradiotherapy for pancreatic cancer.

PurposeMuch attention has been paid to preoperative treatment as a new strategy especially for borderline resectable pancreatic cancer (BRPC). The purpose of this study was to define the optimal indication of neoadjuvant chemoradiotherapy (NACRT) for pancreatic cancer.MethodsWe analyzed consecutive 184 patients who had undergone pancreatic resection in Nara Medical University Hospital. Resectability status was classified by NCCN guidelines. Full-dose gemcitabine with concurrent radiation was used as NACRT. We evaluated 85 patients treated with NACRT in comparison with 99 patients without NACRT as control.ResultsThe regimen of NACRT was well tolerated and feasible. The perioperative outcomes were almost comparable. The postoperative complications were significantly less frequent in NACRT group than non-NACRT group. The pathological effects on both resectable and borderline tumors were favorable in NACRT group compared to non-NACRT group. The overall survival of resectable pancreatic cancer was significantly better than that of BRPC regardless of whether the patients were treated with or without NACRT. The prognosis of the patients with NACRT in resectable tumors was significantly better than without, while there was no significant difference in BRPC. Furthermore, multivariate analysis of various factors in the patients with NACRT identified resectability status and completion of adjuvant chemotherapy as independent prognostic factors.ConclusionsNACRT did not improve the prognosis of the patients with BRPC, although it induced substantial pathological antitumor effect. In contrast, the prognosis of resectable pancreatic cancer treated with NACRT was favorable. Therefore, resectable pancreatic cancer may be good indication for multimodal treatment including NACRT.

Heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinomas with different status of p53

To examine p53-dependency in hyperthermic cancer therapy, heat-induced growth inhibition and apoptosis in transplanted human head and neck squamous cell carcinoma (HNSCC) tumours were analysed with different status of p53 into nude mice. The tumour tissue from HNSCC cell line (SAS) transfected with mutant p53 gene (SAS/mp53) or control vector containing neo gene (SAS/neo) was transplanted into the subcutaneous tissue of the thigh of nude mice using a trocar. Hyperthermia was performed at 42°C when the mean diameter of tumour was 5–6 mm. The diameter of tumours was measured using vernier calipers and tumour weight (TW) and the relative tumour weight (RW) was calculated. Tumour regrowth delay was evaluated when the RW reached 5-fold against the control group. The accumulation of p53 and Bax proteins was examined by an immunohistochemical technique. Apoptotic cells in the sections were detected by staining of DNA ends using an immunohistochemical technique. SAS/mp53 tumours showed more heat-resistance than SAS/neo tumours. The p53-positively staining cells were observed in untreated SAS/mp53 tumours, but not in untreated SAS/neo tumours. After heat treatment, the accumulation of p53 and Bax proteins was observed in SAS/neo tumours, but little in SAS/mp53 tumours. The incidence of apoptotic cells induced by heat treatment was very low in SAS/mp53 tumours compared with SAS/neo tumours. In conclusion, the heat-induced growth inhibition of a transplanted HNSCC may be correlated with the induction of p53-dependent Bax-mediated apoptosis. Thus, p53 status appears to be one of the useful parameters for the predictive assays in hyperthermic cancer therapy.

Arterial Infusion of 5-Fluorouracil Combined with Concurrent Radiotherapy for Unresectable Pancreatic Cancer: Results from a Pilot Study

OBJECTIVE Systemic chemotherapy and chemoradiotherapy, the standard treatments, do not satisfactorily improve the poor prognosis of unresectable pancreatic cancer. The authors administered arterial infusion of 5-fluorouracil (5-FU) combined with concurrent radiation therapy to enhance the antitumor effect of chemotherapy. The purpose of this study was to examine the efficacy and safety of this combined therapy. MATERIALS AND METHODS One or two catheters were placed into the pancreas-supplying arteries angiographically. To obtain adequate drug distribution, the positions of the catheters were determined in accordance with the results of CT during arterial injection of contrast material. A dose of 333 mg/m2/d of 5-FU was continuously infused for 5 days a week for 5 weeks, with concurrent radiation therapy (50 Gy at 2.0 Gy per fraction). Twenty patients with unresectable pancreatic cancer were enrolled in this study. RESULTS Of the 20 patients, 19 (95%) completed the scheduled course of this combined therapy. Fourteen patients showed a partial response (response rate, 70%). Serum cancer antigen 19-9 (CA 19-9) levels were reduced by more than 50% in 16 of 18 patients (80%). The 1-year and 3-year survival rates were 40% and 17%, respectively, with a median survival time of 11.0 months. Grade 3 or worse nonhematologic toxicity was observed in 11 patients (55%), but there were no life-threatening toxicities or complications. CONCLUSION Arterial infusion of 5-FU combined with concurrent radiation therapy is tolerable and can produce a high response rate with encouraging survival duration for unresectable pancreatic cancer.

Importance of resectability status in neoadjuvant treatment for pancreatic cancer

Much attention has been paid to neoadjuvant treatment (NAT) as a new strategy especially for borderline resectable pancreatic cancer (BRPC). However, the optimal indication of NAT remains undetermined.