Low-dose hyperradiosensitivity of human glioblastoma cell lines in vitro does not translate into improved outcome of ultrafractionated radiotherapy in vivo

Purpose: Low dose hyperradiosensitivity (HRS) has been observed in HGL21- and T98G human glioblastoma cells in vitro. The present study investigates whether these effects translate into improved outcome of ultrafractionated irradiation (UF) in vivo.

Material and methods: T98G or HGL21 were transplanted on the hind leg of nude mice. Tumours were irradiated with UF (3 fractions of 0.4 Gy per day, interval 4 h, 7 days per week) or with conventional fractionation (CF; 1 fraction of 1.68 Gy per day, 5 days per week) over 2 or 4 weeks in HGL21 and 2,4 or 6 weeks in T98G. In HGL21, graded top-up doses under clamped hypoxia were applied after 4 weeks of fractionated irradiation. Additional groups of animals were irradiated with single doses under clamp hypoxic conditions with or without whole body irradiation (WBI) before tumour transplantation. Experimental endpoints were growth delay (time to 5-fold starting volume, GD_V5) and local tumour control.

Results: In T98G tumours median relative GD_V5 was 1.2 [95%C.I. 0.96; ∞] in the CF and 0.8 [0.7; 1.02] in the UF arm (p = 0.009) indicating that ultrafractionation is less efficient than conventional fractionation. The TCD₅₀ value of 33.5 Gy [22; 45] after UF was higher than TCD₅₀ of 23.6 Gy [16; 31] after CF (p = 0.15). In HGL21 the median relative GD_V5 was not significantly different between CF and UF. The top-up TCD₅₀ value of 16.1 Gy [95% C.I. 9; 23 Gy] after CF was significantly lower than the corresponding value of 33.2 Gy [23; 44] after UF irradiation (p = 0.007), indicating a higher efficacy of CF compared to UF.

Conclusion: The results on human T98G and HGL21 glioblastoma do not support the hypothesis that HRS in vitro translates into improved outcome of ultrafractionated irradiation in vivo.     

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines

Purpose: To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure.

Materials and methods: Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells.

Results: We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1?Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5?Gy.

Conclusion: These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.     

In vivo uptake of [11C]choline does not correlate with cell proliferation in human prostate cancer

Purpose Prostate cancer is the second leading cause of death from cancer among US men. Positron emission tomography (PET) with [¹¹C]choline has been shown to be useful in the staging and detection of prostate cancer. The background of the increased uptake of choline in human prostate cancer is not completely understood. The aim of this study was to prospectively investigate the relationship between the [¹¹C]choline uptake and the cell proliferation in human prostate cancer.Methods Prostate cancer tissue from 18 patients who had undergone a radical prostatectomy for histologically proven disease was studied. An [¹¹C]choline PET scan was performed prior to surgery. Post-prostatectomy specimens were prepared and stained with the antibody MIB-1 for Ki-67, which depicts proliferation. Two independent observers counted the amount of stained nuclei per specimen. Results Prostate cancer showed Ki-67 staining and high uptake of [¹¹C]choline. Statistical analysis showed no significant correlation between [¹¹C]choline uptake and Ki-67 staining (R=0.23; P=0.34). No significant relationships were found between the uptake of [¹¹C]choline (SUV) and either preoperative PSA (R=0.14; P=0.55) or Gleason sum score (R=0.28; P=0.25).Conclusion In vivo uptake of [¹¹C]choline does not correlate with cell proliferation in human prostate cancer as depicted by Ki-67. Our results suggest that a process other than proliferation is responsible for the uptake of [¹¹C]choline in prostate cancer.     

Field-in-field plan does not improve the dosimetric outcome compared with the wedged beams plan for breast cancer radiotherapy

To evaluate and compare the dosimetry of field-in-field (FIF) and wedged beams (WB) techniques for patients with breast cancer receiving adjuvant radiotherapy after conservative surgery. A total of 89 patients with breast cancer participated in this study. Each patient received a computed tomography–based treatment plan with opposed tangential fields. Two planning techniques (FIF and WB) were generated for each patient by using the Pinnacle treatment-planning system. Three indices, the homogeneity index (HI), conformity index (CI), and uniformity index (UI), as well as maximum dose (D_max), median dose (D₅₀), number of portals, monitor unit (MU), and lung volume at 20 Gy (lung₂₀) were used for comparison. The mean values tested using a t-test indicated that the WB technique had a significantly lower HI (p < 0.0001), a significantly higher CI (p < 0.0001), and a significantly higher D₅₀ (p = 0.0002) than did the FIF technique. The FIF technique had a significantly higher D_max compared with the WB technique, but lung₂₀ did not exhibit a significant difference. By contrast, the FIF technique had a significantly higher UI and a significantly lower MU compared with the WB technique, but a significantly higher number of portals were found in the FIF technique. The FIF technique did not demonstrate superior dosimetric results. The WB technique had a significantly lower HI, higher CI, lower D_max, and lower number of portals; but the FIF technique had a significantly higher UI and lower MU.     

Unlabelled iododeoxyuridine increases the cytotoxicity and incorporation of [1251]-iododeoxyuridine in two human glioblastoma cell lines

Iododeoxyuridine (IUdR), labelled with radioiodines emitting Auger, alpha or beta- radiation, has been proposed as a therapeutic tool in the treatment of cancer. However, the low per cent incorporation in tumour cells and limited cytotoxicity are major obstacles for such an application. Using unlabelled IUdR as a modulator, we have studied the in vitro cytotoxicity of [125I]-IUdR in two human glioblastoma cell lines. Surprisingly, an enhanced cytotoxicity of [125I]-IUdR was observed in the presence of 0.3-10 microM concentrations of unlabelled IUdR in U251 glioblastoma cells and to a lesser extent in LN229 cells. The presence of unlabelled IUdR unexpectedly increased the incorporation of [125I]-IUdR in both cell lines. Thymidine competitively blocked the cytotoxic effects of combined unlabelled and [125I]-labelled IUdR in these cells and DNA-incorporation of radiolabelled IUdR.     

人非小细胞肺癌细胞受照射后肿瘤坏死因子水平的变化

目的观察非小细胞肺癌(NSCLC)细胞系A549和NCI-H596受x射线照射后肿瘤坏死因子(TNF-α)mRNA表达及其蛋白水平的变化。方法应用实时荧光RT-PCR技术，观察肺癌细胞系A549和NCI-H596照射2、5、10、20、30和40Gy后1、3、6、12、24、48和72hTNF．amRNA表达及其蛋白水平。应用免疫组织化学法(EusA)检测相应培养液中TNF-α蛋白的水平。同时进行集落形成实验观察2种细胞系的放射敏感性。结果X射线照射后TNF-α mRNA表达及其蛋白水平较照射前升高，吸收剂量达40q时TNF-α mRNA表达水平达高峰，为对照组的83倍。NCI-H596细胞受照射后1h TNF-α mRNA表达逐渐升高，6h达高峰，为对照组的568倍。NCI-H596细胞受照射后TNF-α蛋白水平逐渐升高，受照射后24h达高峰(为基础水平的58倍)。且与受照射剂量有关。A549细胞受照射后1h TNF-α mRNA表达仅见瞬间升高，随后即降至基础水平。而且，A549细胞受照射后TNF-α蛋白水平无明显升高。结论X射线可诱导NSCLC细胞系NCI-H596产生TNF-α，呈现时间、剂量依赖性改变。     

Ionizing radiation induces up-regulation of functional beta1-integrin in human lung tumour cell lines in vitro

PURPOSE: Cell-matrix interactions are in part mediated through the beta1-integrin pathway regulating cell survival, proliferation, adhesion and migration. This study was performed to elucidate alterations of expression of the beta1-integrin and its co-localized protein kinase, integrin-linked kinase (ILK), after exposure to ionizing radiation in two lung carcinoma cell lines in the presence or absence of different beta1-integrin-dependent matrix proteins. MATERIALS AND METHODS: Exponentially growing A549 and SKMES1 cells grown on fibronectin, laminin, BSA or plastic were exposed to 2 Gy or 6 Gy. Besides colony formation assays (0.5-8 Gy) and immediate plating experiments, flow cytometry (for beta1-integrin) and immunoblotting (for beta1-integrin and ILK) were carried out to analyze the protein expression. The localization of both proteins plus filamentous (f-) actin was further examined by immunofluorescence staining and laser confocal scanning microscopy. Functionality of the beta1 receptor subunit after irradiation was investigated in adhesion assays. RESULTS: A549 and SKMES1 cells grown on fibronectin or laminin demonstrated a significant increase in cell survival after irradiation compared to cells grown on BSA or plastic. Immediate plating of cells after irradiation on fibronectin did not show an improved survival. Flow cytometric and Western blot data showed a dose- and matrix-dependent induction of beta1-integrin and ILK expression after irradiation within 48 h. Adhesion to fibronectin or laminin compared to BSA or plastic was increased by 10-fold after irradiation, demonstrating these specific cell surface receptors to be functional. The staining of beta1-integrin and ILK in A549 cells confirmed the radiation-induced up-regulation of both proteins. Additionally, beta1-integrin and ILK co-localized with accumulated actin fibers at the cytoplasmic face of the cell membrane at confined areas. CONCLUSIONS: Ionizing radiation strongly induced the expression of functional beta1-integrin and ILK in the two lung cancer cell lines, A549 and SKMES1, dependent on different matrices used. Additionally, the subcellular localization of both proteins was altered by irradiation, and the individual cellular radiosensitivity was reduced in the presence of an extracellular matrix. On the one hand, this may result in aggravated therapeutic approaches and on the other hand, cells could adhere more strongly in their environment by the increase in functional surface receptor density preventing metastasis. Concerning intravascular located tumour cells, beta1-integrin up-regulation might enable these cells to adhere to the endothelium, which represents a prerequisite for metastatic disease. Identification of such mechanisms will provide considerable insights into the understanding of tumorigenicity and metastatic phenotypes, possibly leading to new, optimized radiochemotherapeutic regimens.     

In vitro and in vivo targeting of different folate receptor-positive cancer cell lines with a novel 99mTc-radiofolate tracer

PURPOSE: For the assessment of folate-based radiopharmaceuticals, human nasopharyngeal KB carcinoma cells are traditionally used although nasopharyngeal cancer is rare. On the other hand, the folate receptor (FR) is frequently overexpressed on diverse cancer types, the highest frequency (>90%) being on ovarian carcinomas. The goal of our study was the in vitro and in vivo assessment of different FR-positive human carcinoma cells. In addition, a murine sarcoma cell line was assessed as a pre-clinical alternative to human xenograft models. METHODS: FR-positive human nasopharyngeal, cervical, ovarian and colorectal cancer cell lines and the transgenic mouse sarcoma (24JK-FBP) cell line were targeted with a novel 99mTc-tricarbonyl folate derivative 2. Comparative in vitro cell binding studies were carried out under standardised folate-deficient conditions. In vivo studies were performed in nude mice and C6 black mice. RESULTS: The in vitro cell experiments revealed only FR-specific binding (unspecific <0.02%), ranging from 3.5% to 52% of complex 2 owing to variable levels of FR expression of the cell lines. In vivo tumour uptake of radiotracer 2 varied less than in vitro. It ranged from 0.66+/-0.17% ID/g (LoVo) through 1.16+/-0.64% ID/g (IGROV-1) and 1.55+/-0.43% ID/g (24JK-FBP) to 2.33+/-0.36% ID/g (KB) 4 h p.i. CONCLUSION: These pre-clinical studies indicate that in vitro data obtained in FR-positive cancer cells do not necessarily correspond with or predict in vivo radiofolate uptake in corresponding (xeno)grafts. In addition, the murine 24JK-FBP cell line proved to be a valuable pre-clinical alternative to human tumour models.     

Effects of lonidamine alone or in combination with radiotherapy on survival and metabolism in human and rodent cell lines]

In vitro investigations on the combined effect of radiation and Lonidamine in one human (LXI) and two rodent cell lines (3T3, V79) showed not a clear radiosensitizing effect. Especially no inhibiting influence on the repair of potentially lethal damage of V79 cells was found. Incubation at lower pH values (6.7 to 6.9) caused no increasing effects. Lonidamine induced damage on the mitochondrial dehydrogenase-system was quantitatively measured with a modified MTT-assay.     

Integration of PET-imaging into radiotherapy treatment planning for low-grade meningiomas improves outcome

European Journal of Nuclear Medicine and Molecular Imaging - Meningiomas have an excellent survival prognosis, and radiotherapy (RT) is a central component of interdisciplinary treatment. During...     

Role of DNA-PK in the process of aberration formation as studied in irradiated human glioblastoma cell lines M059K and M059J

PURPOSE: The participation of various DNA double-strand break repair mechanisms in the formation of chromosome aberrations is not yet fully understood. To investigate particularly the role of non-homologous end-joining, we analysed the formation of radiation-induced aberrations in a DNA-protein kinase (PK(CS))-proficient cell line M059K and in a deficient cell line M059J. MATERIALS AND METHODS: Plateau-phase M059K and M059J cells were irradiated with low doses of X-rays. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations and total visible complex exchanges (complex aberrations) for chromosomes 4, 7 and 11 using the FISH method. M059K cells were also analysed in the presence of 50 micro m wortmannin, a DNA-PK inhibitor. RESULTS: DNA-PK-deficient cells showed a higher yield of simple stable and unstable and of complex aberrations in comparison with DNA-PK-proficient cells. The largest differences were observed for excess acentric fragments and for complex aberrations. DNA-PK inhibition by wortmannin in M059-K cells resulted in increased aberration yield in the same qualitative and quantitative manner as in M059-J cells. CONCLUSIONS: The results obtained with DNA-PK-deficient M059J cells and with DNA-PK-proficient M059K cells treated with wortmannin, an inhibitor of DNA-PK and ATM, suggest that the elimination of DNA-PK-dependent non-homologous end-joining can recruit a slow, error-prone repair process, which is DNA-PK independent and favours the increased formation of chromosome aberrations. The nature of this pathway and the way of its participation in aberration formation need further elucidation.     

p53 status,cellular recovery and cell cycle arrest as prognosticators of in vitro radiosensitivity in human pancreatic adenocarcinoma cell lines

Purpose: To investigate the factors contributing to the in vitro radiosensitivity of four human pancreatic adenocarcinoma cell lines differing in p53 status, and the basis for the lack of post-irradiation G1 arrest in the two cell lines that have retained a wild-type p53 allele. Materials and methods: Cells were X-irradiated and the parameters related to radiosensitivity, as well as the modulation of gene products linked to regulation of cell cycle transit (p53, p21/WAF1/CIP1, pRb) or DNA replication and repair (DNA topoisomerase I and II), were determined. Results: Both cell lines expressing either mutant (mt) R248W or R273H p53 proteins were more radioresistant. All the cell lines arrested in G2. None of the cell lines arrested in G1 and this was linked to the inability to upregulate p21/WAF1/CIP1. There were no correlations between p53 status and the magnitude or time of maximum G2 arrest. However, there was a negative correlation between a protracted arrest in G2 and the ability to recover from potentially lethal damage (PLD). Conclusions: Variation in radiosensitivity is related to p53 status, but the survival advantage conferred by having mutant p53 status is not readily explained neither by recovery from PLD nor by cell cycle arrest kinetics. There is no p53-independent pathway for the recruitment of p21 in these cell lines following irradiation.     

p53 status, cellular recovery and cell cycle arrest as prognosticators of in vitro radiosensitivity in human pancreatic adenocarcinoma cell lines

PURPOSE: To investigate the factors contributing to the in vitro radiosensitivity of four human pancreatic adenocarcinoma cell lines differing in p53 status, and the basis for the lack of post-irradiation G1 arrest in the two cell lines that have retained a wild-type p53 allele. MATERIALS AND METHODS: Cells were X-irradiated and the parameters related to radiosensitivity, as well as the modulation of gene products linked to regulation of cell cycle transit (p53, p21/WAF1/CIP1, pRb) or DNA replication and repair (DNA topoisomerase I and II), were determined. RESULTS: Both cell lines expressing either mutant (mt) R248W or R273H p53 proteins were more radioresistant. All the cell lines arrested in G2. None of the cell lines arrested in G1 and this was linked to the inability to upregulate p21/WAF1/CIP1. There were no correlations between p53 status and the magnitude or time of maximum G2 arrest. However, there was a negative correlation between a protracted arrest in G2 and the ability to recover from potentially lethal damage (PLD). CONCLUSIONS: Variation in radiosensitivity is related to p53 status, but the survival advantage conferred by having mutant p53 status is not readily explained neither by recovery from PLD nor by cell cycle arrest kinetics. There is no p53-independent pathway for the recruitment of p21 in these cell lines following irradiation.     

In vitro and in vivo tracer characteristics of an established multidrug-resistant human colon cancer cell line.

99mTc-methoxyisobutylisonitrile (99mTc-MIBI) has been suggested as a tracer for the scintigraphic detection of multidrug resistance (MDR). The aim of this study was to compare MDR characteristics in vitro and in vivo by immunohistochemic and functional uptake assays in established tumor cell lines cultured and grown in severe combined immunodeficient (SCID) mice. METHODS: The presence of MDR was assessed in vitro in drug-resistant HT-29(mdr1) colon carcinoma cells and in nonresistant HT-29(par) cells by JSB-1 immunohistochemistry, uptake of the fluorescent dye Rhodamine 123, and quantitative measurement of 99mTc-MIBI accumulation. For in vivo imaging, SCID mice bearing subcutaneous xenografts of these cell lines were injected with 99mTc-MIBI and 18F-FDG for scintigraphic and PET examination. After imaging, tumors were analyzed by immunohistochemistry and electron microscopy. RESULTS: All HT-29(mdr1) cells cultured in vitro exhibited distinct JSB-1 immunoreactivity, although to a variable degree, whereas HT-29(par) cells were completely devoid of JSB-1 staining. Rhodamine 123 accumulated poorly in HT-29(mdr1) cells but strongly in HT-29(par) cells. Accumulation of 99mTc-MIBI was 0.05% +/- 0.01% of the activity of the external medium in HT-29(mdr1) cells, but about eight times higher in HT-29(par) cells (0.40% +/- 0.09%), a very low percentage compared with other tumor cell lines. No difference in 201TlCl accumulation was observed between both cell lines. In vivo, neither HT-29(par) nor HT-29(mdr1) tumors grown in SCID mice could be detected by 99mTc-MIBI scintigraphy. In FDG PET, both HT-29(mdr1) and HT-29(par) tumors were clearly visible. FDG uptake was, however, markedly higher in HT-29(par) than in HT-29(mdr1) tumors. Both tumor types were poorly vascularized, as shown histologically. JSB-1 immunoreactivity was absent in all HT-29(par) tumors examined, whereas the majority of HT-29(par) tumor cells were stained. Electron microscopy showed that HT-29(par) tumors contained significantly less mitochondria than hepatocytes of the SCID mouse liver, which displayed high 99mTc-MIBI uptake in our scintigraphy studies. CONCLUSION: Sufficient 99mTc-MIBI uptake is the major prerequisite for distinguishing successfully between drug-resistant and sensitive cells. Negative 99mTc-MIBI scintigrams are not necessarily associated with MDR expression. In some tumors, FDG may be an in vivo marker for MDR as suggested by PET.     

Radionuclide angiography with technetium-99m in vivo labeled erythrocytes does not lead to induction of mutations in the HPRT gene of human T-lymphocytes

Mutant frequencies were measured in T-lymphocytes of patients undergoing radionuclide angiography with erythrocytes labeled in vivo with technetium-99m. Blood from 13 patients was sampled before and after (8-120 days) an injection with 750 MBq technetium-99m. Frequencies of HPRT- mutants were measured with the T-cell cloning method. Results indicated that the mean frequency of mutants after treatment was significantly below that measured before exposure. Thus, in contrast to published data, our results do not support the conclusion that radionuclide angiography with technetium-99m induces HPRT- mutations. Further analysis of our data indicated that the decrease in mutant frequency after exposure can be accounted for by an effect of cloning efficiency.     

Ultrasound-triggered microbubble destruction enhances the radiosensitivity of glioblastoma by inhibiting PGRMC1-mediated autophagy in vitro and in vivo

Background: Ultrasound-triggered microbubble destruction(UTMD) is a widely used noninvasive technology in both military and civilian medicine, which could enhance radiosensitivity of various tumors. However, little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism. This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.Methods: GL261, U251 cells and ortho...