Efficient uptake and retention of iron oxide-based nanoparticles in HeLa cells leads to an effective intracellular delivery of doxorubicin.

The purpose of this study was to construct and characterize iron oxide nanoparticles (IONPCO) for intracellular delivery of the anthracycline doxorubicin (DOX; IONPDOX) in order to induce tumor cell inactivation. More than 80% of the loaded drug was released from IONPDOX within 24 h (100% at 70 h). Efficient internalization of IONPDOX and IONPCO in HeLa cells occurred through pino- and endocytosis, with both IONP accumulating in a perinuclear pattern. IONPCO were biocompatible with maximum 27.9% ± 6.1% reduction in proliferation 96 h after treatment with up to 200 µg/mL IONPCO. Treatment with IONPDOX resulted in a concentration- and time-dependent decrease in cell proliferation (IC50 = 27.5 ± 12.0 µg/mL after 96 h) and a reduced clonogenic survival (surviving fraction, SF = 0.56 ± 0.14; versus IONPCO (SF = 1.07 ± 0.38)). Both IONP constructs were efficiently internalized and retained in the cells, and IONPDOX efficiently delivered DOX resulting in increased cell death vs IONPCO.

Adaptive fractionated stereotactic Gamma Knife radiotherapy of meningioma using integrated stereotactic cone-beam-CT and adaptive re-planning (a-gkFSRT).

OBJECTIVE: The Gamma Knife Icon (Elekta AB, Stockholm, Sweden) allows frameless stereotactic treatment using a combination of cone beam computer tomography (CBCT), a thermoplastic mask system, and an infrared-based high-definition motion management (HDMM) camera system for patient tracking during treatment. We report on the first patient with meningioma at the left petrous bone treated with adaptive fractionated stereotactic radiotherapy (a-gkFSRT). METHODS: The first patient treated with Gamma Knife Icon at our institute received MR imaging for preplanning before treatment. For each treatment fraction, a daily CBCT was performed to verify the actual scull/tumor position. The system automatically adapted the planned shot positions to the daily position and recalculated the dose distribution (online adaptive planning). During treatment, the HDMM system recorded the intrafractional patient motion. Furthermore, the required times were recorded to define a clinical treatment slot. RESULTS: Total treatment time was around 20 min. Patient positioning needed 0.8 min, CBCT positioning plus acquisition 1.65 min, CT data processing and adaptive planning 2.66 min, and treatment 15.6 min. The differences for the five daily CBCTs compared to the reference are for rotation: -0.59 ± 0.49°/0.18 ± 0.20°/0.05 ± 0.36° and for translation: 0.94 ± 0.52 mm/-0.08 ± 0.08 mm/-1.13 ± 0.89 mm. Over all fractions, an intrafractional movement of 0.13 ± 0.04 mm was observed. CONCLUSION: The Gamma Knife Icon allows combining the accuracy of the stereotactic Gamma Knife system with the flexibility of fractionated treatment with the mask system and CBCT. Furthermore, the Icon system introduces a new online patient tracking system to the clinical routine. The interfractional accuracy of patient positioning was controlled with a thermoplastic mask and CBCT.

Endothelial Caveolin-1 regulates the radiation response of epithelial prostate tumors.

The membrane protein caveolin-1 (Cav1) recently emerged as a novel oncogene involved in prostate cancer progression with opposed regulation in epithelial tumor cells and the tumor stroma. Here we examined the role of stromal Cav1 for growth and radiation response of MPR31-4 prostate cancer xenograft tumors using Cav1-deficient C57Bl/6 mice. Syngeneic MPR31-4 tumors grew faster when implanted into Cav1-deficient mice. Increased tumor growth on Cav1-deficient mice was linked to decreased integration of smooth muscle cells into the wall of newly formed blood vessels and thus with a less stabilized vessel phenotype compared with tumors from Cav1 wild-type animals. However, tumor growth delay of MPR31-4 tumors grown on Cav1 knockout mice to a single high-dose irradiation with 20 Gray was more pronounced compared with tumors grown on wild-type mice. Increased radiation-induced tumor growth delay in Cav1-deficient mice was associated with an increased endothelial cell apoptosis. In vitro studies using cultured endothelial cells (ECs) confirmed that the loss of Cav1 expression increases sensitivity of ECs to radiation-induced apoptosis and reduces their clonogenic survival after irradiation. Immunohistochemical analysis of human tissue specimen further revealed that although Cav1 expression is mostly reduced in the tumor stroma of advanced and metastatic prostate cancer, the vascular compartment still expresses high levels of Cav1. In conclusion, the radiation response of MPR31-4 prostate tumors is critically regulated by Cav1 expression in the tumor vasculature. Thus, Cav1 might be a promising therapeutic target for combinatorial therapies to counteract radiation resistance of prostate cancer at the level of the tumor vasculature.

Changes in telomerase activity after irradiation of human peripheral blood mononuclear cells (PBMC) in vitro.

Telomerase activity (TA) has been shown to be up-regulated by ionising radiation in immortal hematopoietic cell lines. The purpose of the present experiments was to test whether ionising radiation may regulate TA in normal human peripheral blood mononuclear cells (PBMC). A real-time PCR assay was established to quantify TA detected by the telomeric repeat amplification protocol. TA of PBMCs isolated from young healthy donors was highly increased by stimulation with phytohemagglutinin (PHA) for 72 h. Irradiation of PHA-stimulated PBMCs with 2-10 Gy of X rays showed up-regulated TA 4 h after irradiation with an enhancement at least as strong as for the human TK6 lymphoblastoid cell line. The present results show that TA is up-regulated by irradiation not only in immortal cell lines but also in mitogen-stimulated PBMCs. This supports a possible role for telomerase in the cellular radiation response.

Influence of different dose rates on cell recovery and RBE at different spatial positions during protracted conformal radiotherapy.

The effect of simultaneous induction and repair of sub-lethal lesions during protracted irradiation is potentially important for conformal irradiation techniques and may influence the relative biological effectiveness (RBE) of different radiation qualities. The importance of repair for 50 kV X rays from a miniaturised X-ray source producing an essentially isotropic dose distribution for intra-operative radiotherapy (IORT) was verified for inactivation of V79 cells in different distances from the source. The experimental data were evaluated in terms of the linear-quadratic model and the generalised Lea-Catcheside time factor. Furthermore, the shape of survival curves for 14 MeV [d(0.25) + T] neutrons at different dose rates implicated a role of repair for fast neutrons. Microdosimetric measurements have previously demonstrated variations in radiation quality between different positions in a therapy phantom for conformal moving-beam therapy with this radiation. Experimental data on the effectiveness for inactivation of V79 cells irradiated at such positions are presented and the influence of repair is analysed.

Spatial variation of radiation quality during moving beam therapy with 14 MeV [d(0.25)+T] neutrons.

In conformal moving beam therapy with fast neutrons, the contributions to dose from the direct beam, scattered radiation and the gamma component vary with the position in the phantom. To determine this variation in radiation quality, microdosimetric measurements of energy deposition spectra were performed at different position in a therapy phantom. Fixed beam irradiation at different incidence angles showed strong changes in the lineal energy spectrum. An increase of slow protons (20 < y < 110 keV.micron-1) and a decrease of fast protons (2 < y < 20 keV.micron-1) was seen for irradiation outside the direct beam. During moving beam irradiation, different positions on the same isodose curves (55% or 35%) showed differences in YD of up to 5%. Variations in the quality parameter, R, determined by applying an empirical biological weighting function, were of similar magnitude. Thus, spatial variations in radiation quality should be taken into account in biological dose planning for moving beam neutron therapy.

Irradiated homozygous TGF-beta1 knockout fibroblasts show enhanced clonogenic survival as compared with TGF-beta1 wild-type fibroblasts.

PURPOSE: To study the role of transforming growth factor beta1 (TGF-beta1) on cellular radiation sensitivity by analysing mouse lung fibroblasts of different TGF-beta1 genotypes. MATERIALS AND METHODS: Heterozygous TGF-beta1 knock-out mice were mated to produce offspring of different TGF-beta1 genotypes as confirmed by PCR-genotyping. Primary lung fibroblast populations were established from new-born animals of specific genotypes (TGF-beta1(+/+), TGF-beta1(+/-), TGF-beta1(-/-)). Production of TGF-beta1 was tested by ELISA. TGF-beta1 receptor-II mRNA expression was analysed by RT-PCR. Colony formation of untreated, TGF-beta1-treated and/or irradiated primary lung fibroblasts was determined under different medium conditions. RESULTS: Plating efficiencies under different medium conditions were independent of TGF-beta1 genotype. Production of TGF-beta1 correlated with the genotype: heterozygous TGF-beta1 knock-out fibroblasts (TGF-beta1(+/-)) produced 60-65% of wild-type (TGF-beta1(+/+) cells). As expected, homozygous TGF-beta1 knock-out fibroblasts (TGF-beta1(-/-)) did not produce TGF-beta1. Radiation exposure significantly enhanced TGF-beta1 production in TGF-beta1(+/+) cells by a factor of 2. No such stimulation was observed in TGF-beta1(+/-) cells. TGF-beta1(+/-) and especially TGF-beta1(-/-) cells were significantly more radioresistant than TGF-beta1(+/+) cells. TGF-beta1 treatment significantly reduced clonogenic survival for both TGF-beta1(+/+) and TGF-beta1(-/-) cells. TGF-beta1 treatment of TGF-beta1(-/-) cells resulted in an enhancement of radiation sensitivity. CONCLUSION: The data are the first direct evidence that TGF-beta1 is a major autocrine regulator of intrinsic radiation sensitivity of mouse lung fibroblasts.

Differential response of tumor cells and normal fibroblasts to fractionated combined treatment with topotecan and ionizing radiation.

PURPOSE: The impact of the topoisomerase-I inhibitor topotecan (Hycamtin) as a single agent or in combination with ionizing radiation on clonogenic cell survival has been evaluated in three tumor cell lines and in normal fibroblasts. Both agents have been applied in a fractionated scheme in order to assess clinically relevant conditions. MATERIALS AND METHODS: Cell inactivation was investigated in human glioblastoma cell lines U118 and U138, lung carcinoma cell line A549 and normal fibroblasts of the skin (HSF6) and lung (CCD32) using the colony formation assay. RESULTS: The glioblastoma cell lines were highly sensitive to the drug, whereas normal fibroblasts were much less sensitive. A significant antagonistic effect of the drug combined with irradiation was found for normal fibroblasts, while glioblastoma cells showed no evidence of interaction, indicating additivity. A549 cells showed a biphasic response to topotecan alone and in combination with irradiation, suggesting induction of resistence to the drug. CONCLUSION: Differential effects of combined topotecan/radiation treatment were detected between cells of normal tissue and tumor cells, being antagonistic in fibroblasts of the skin and lung, but not in tumor tissue, especially in glioblastoma cells.

Spontaneous and radiation-induced differentiationof fibroblasts.

Clonal heterogeneity in fibroblast cultures from donors of all ages has been associated with differentiation of the fibroblast/fibrocyte system. Thus, a terminal differentiation lineage including a sequence of three potentially mitotic progenitor fibroblasts (MFI-->MFII-->MFIII) in the precursor compartment and three types of postmitotic fibrocytes (PMFIV-->PMFV-->PMFVI) in the functional compartment has been identified previously. In the present study, we show that replenishment of fibrocytes lost from the functional compartment is not expected to change the distribution of differentiation types in a steady state population, provided cell loss occurs at the end of a long sequence of cell divisions only. However, premature terminal differentiation of progenitor fibroblasts to postmitotic fibrocytes can be induced by ionising radiation and other cell stressors. Furthermore, even a low dose of 1Gy causes a change in the distribution of surviving MF progenitor cells towards later differentiation stages within the precursor compartment. The role of autocrine transforming growth factor-beta1 production by fibroblasts in mediating terminal differentiation was investigated. We propose that cell stress and DNA damaging agents may contribute to progression of the differentiation state with age and that individual variation may be related to differences in the rate of induced differentiation.

Fibroblast differentiation in subcutaneous fibrosis after postmastectomy radiotherapy.

In order to acquire a better understanding of the mechanism of radiation-induced fibrosis, we studied the differentiation of normal skin fibroblasts cultured from breast cancer radiotherapy patients with different risk of fibrosis. The differentiation state of fibroblasts was characterized in clonal cultures using established cytomorphological criteria. Collagen synthesis was determined by 3H-proline incorporation into pepsin-resistant protein. Radiation-induced inactivation of fibroblasts was paralleled by an increase in terminally differentiated fibrocytes, demonstrating that premature terminal differentiation is an important response to irradiation of fibroblasts from radiotherapy patients. Surviving colony-forming fibroblasts showed a change in differentiation with an increase in the ratio L:E of progenitor fibroblasts in late (L) compared to early (E) differentiation states. Furthermore, increased collagen production was observed after irradiation. The results provide evidence supporting a role of terminal fibroblast differentiation in radiation-induced fibrosis and imply that the progenitor population surviving radiotherapy might be more prone to terminal differentiation than before radiotherapy.

Radiation sensitivity of human squamous cell carcinoma cells in vitro is modulated by all-trans and 13-cis-retinoic acid in combination with interferon-alpha.

PURPOSE: Retinoids and interferon-alpha (IFN-alpha) have been shown to exert antiproliferative and radiosensitizing effects. The present study was designed to determine differential effects of retinoids in combination with IFN-alpha on radiation toxicity of 5 human squamous cell carcinoma (SCC) cell lines. METHODS AND MATERIALS: Using clonogenic assays, the effects of all-trans (ATRA), 13-cis-retinoic acid (13cRA), and IFN-alpha on radiation toxicity were analyzed. Basal mRNA expression of the cytoplasmic retinoic acid binding protein, CRABP I, was determined in retinoid-sensitive and -insensitive cell lines by reverse transcriptase/polymerase chain reaction (RT-PCR). RESULTS: Treatment with ATRA, 13cRA, or IFN-alpha resulted in a cell line-specific inhibition of clonogenic survival. A comparison of retinoid-sensitive and insensitive cells revealed that retinoid sensitivity seems to be dependent on the basal expression level of CRABP I. ATRA, 13cRA, and IFN-alpha alone or in combination altered radiation sensitivity by affecting predominantly the alpha-component of the linear-quadratic dose-response curve. Likewise, depending upon the treatment condition the surviving fraction at 2 Gy (SF2) was decreased cell line-specifically. Combined treatment with ATRA or 13cRA and IFN-alpha markedly enhanced radiation cytotoxicity. CONCLUSION: These in vitro data indicate that the combined treatment with retinoids, IFN-alpha, and ionizing radiation could be beneficial for patients presenting with SCC.

The role of cytokines in the development of normal-tissue reactions after radiotherapy.

BACKGROUND: Cytokines are important for signaling between cells and tissues and constitute a humoral component of the response of cells and tissues to radiotherapy. Although several cytokines have been implicated in mediating radiation-induced reactions of normal tissues, the mechanisms are only beginning to be elucidated. METHODS: Published data on radiation-induced cytokine expression from cell culture, experimental and clinical studies are reviewed. Current models of cytokine-mediated multicellular interactions in radiation-induced reactions of the lung, skin and intestinal mucosa are presented. RESULTS AND CONCLUSION: The major cytokines in the radiation response of non-hemopoietic tissues include IL-6, IL-1, INF alpha and TGF beta. Different cell types interact via cytokines in a complex network of effector and receptor cells, including inflammatory cells, tissue-specific functional cells and fibroblasts. TGF beta appears to be of particular importance in the development of late reactions such as fibrosis.

Differentiation state of skin fibroblast cultures versus risk of subcutaneous fibrosis after radiotherapy.

BACKGROUND AND PURPOSE: There is increasing evidence for patient-to-patient variation in the response of normal tissue to radiotherapy. Recently, it has been suggested that accumulation of functional fibrocytes may be a key step in the development of radiation-induced fibrosis. Therefore, we have examined a possible relationship between the differentiation state of untreated fibroblasts and the risk of radiation-induced subcutaneous fibrosis in individual patients. MATERIALS AND METHODS: We used skin fibroblast cultures isolated from eight postmastectomy radiotherapy patients whose individual clinical radiosensitivity was assessed by the mean excess risk of fibrosis. Different types of potentially mitotic progenitor fibroblasts (MF) and postmitotic functional fibrocytes (PMF) in the terminal differentiation lineage, MFI --> MFII --> MFIII --> PMF, were scored morphologically in clonal culture. Progression of differentiation was quantified by the ratio L/E of colony-forming late (MFIII and late MFII) and early (MFI and early MFII) progenitors. RESULTS: We observed a correlation between the ratio L/E and the mean risk of fibrosis (rs = 0.743, P = 0.03), indicating an approximately 10-fold increase in L/E with an increasing risk of fibrosis. This was paralleled by a decreasing trend in the absolute numbers of early progenitor types. By contrast, there was no significant correlation between the plating efficiency and the risk of fibrosis. CONCLUSIONS: The data suggest that the risk of fibrosis increases with the progression of the differentiation of untreated progenitor fibroblasts, indicating that the progression of fibroblast differentiation may be a co-factor in the development of radiation-induced fibrosis. If this hypothesis is validated, it provides a rationale for a novel predictive test to identify patients with an increased risk of subcutaneous fibrosis.

Changes in RBE of 14-MeV (d + T) neutrons for V79 cells irradiated in air and in a phantom: is RBE enhanced near the surface?

PURPOSE: The relative biological effectiveness (RBE) for inactivation of V79 cells was determined as function of dose at the Heidelberg 14-MeV (d + T) neutron therapy facility after irradiation with single doses in air and at different depths in a therapy phantom. Furthermore, to assess the reproducibility of RBE determinations in different experiments we examined the relationship between the interexperimental variation in radiosensitivity towards neutrons with that towards low LET 60Co photons. METHODS: Clonogenic survival of V79 cells was determined using the colony formation assay. The cells were irradiated in suspension in small volumes (1.2 ml) free in air or at defined positions in the perspex phantom. Neutron doses were in the range, Dt = 0.5-4 Gy. 60Co photons were used as reference radiation. RESULTS: The radiosensitivity towards neutrons varied considerably less between individual experiments than that towards photons and also less than RBE. However, the mean sensitivity of different series was relatively constant. RBE increased with decreasing dose per fraction from RBE = 2.3 at 4 Gy to RBE = 3.1 at 0.5 Gy. No significant difference in RBE could be detected between irradiation at 1.6 cm and 9.4 cm depth in the phantom. However, an approximately 20% higher RBE was found for irradiation free in air compared with inside the phantom. Combining the two effects, irradiation with 0.5 Gy free in air yielded an approximately 40% higher RBE than a dose of 2 Gy inside the phantom. CONCLUSION: The measured values of RBE as function of dose per fraction within the phantom is consistent with the energy of the neutron beam. The increased RBE free in air, however, is greater than expected from microdosimetric parameters of the beam and may be due to slow recoil protons produced by interaction of multiply scattered neutrons or to an increased contribution of alpha particles from C(n, alpha) reactions near the surface. An enhanced RBE in subcutaneous layers of skin combined with an increase in RBE at low doses per fraction outside the target volume could potentially have significant consequences for normal tissue reactions in radiotherapy patients treated with fast neutrons.

Growth-state-dependent radiation-induced expression of the proto-oncogene c-fos in NIH 3T3 cells.

Expression of the proto-oncogene c-fos in response to ionizing radiation has been observed in some but not all cell lines tested. Here we report on delayed, transient c-fos expression in NIH 3T3 cells induced by 60Co gamma rays in the dose range 2-5 Gy. Induction of c-fos was significantly increased in cells irradiated in the density-arrested quiescent state compared to irradiation in the exponential growth phase. The enhancement correlated with the transition to quiescence as measured by the proliferation markers, proliferating cell nuclear antigen and bromodeoxyuridine. The observation of growth-state-dependent expression of c-fos after irradiation might indicate a functional relationship between c-fos and growth control in the DNA damage response, e.g. a potential role of c-fos in the control of replicative proteins.

Sulfhydryl protection and the oxygen effect on radiation-induced inactivation of r-chromatin in vitro. Influence of an OH scavenger: t-butanol.

Transcriptionally active r-chromatin from Tetrahymena has been irradiated in dilute phosphate buffer, pH 7.2, in the presence of the sulfhydryl compound 2-mercaptoethanol [MSH]. MSH was more protective against radiation-induced inactivation of transcription under N2 than under O2. The OH scavenger, t-butanol, on the other hand, gives significantly less protection under N2 than under O2, apparently due to inactivation by secondary t-butanol radicals under anoxia as shown previously (C. Herskind and O. Westergaard, Radiat. Res., 114, 28-41 (1988). However, MSH was found to restore most of the protective effect of t-butanol under N2. Inactivation was studied as a function of MSH concentration [0.03-10 mM] at different, fixed concentrations of t-butanol [3-300 mM]. The observed protection may be explained essentially in terms of (1) OH scavenging, (2) "repair" of DNA radicals by H-atom transfer from MSH under N2 in competition with fixation of damage under O2, and (3) protection against inactivation by secondary t-butanol radicals by H-atom transfer to these radicals. The sensitizing effect of oxygen in the presence of MSH is reduced by t-butanol and may even be reversed to produce an apparently protective effect. This finding is discussed in terms of residual inactivation by secondary radicals. The significance of OH scavengers as potential modifiers of oxygen enhancement ratio values is discussed.

Variable protection by OH scavengers against radiation-induced inactivation of isolated transcriptionally active chromatin: the influence of secondary radicals.

Isolated r-chromatin, the chromatin form of the extrachromosomal gene coding for the rRNA precursor in Tetrahymena, has been used to study radiation-induced inactivation in vitro in the presence of the OH radical scavengers, t-butanol, formate ions, and methanol. Induction of biologically important DNA lesions was detected by the effect on transcription by endogenous RNA polymerases associated with the isolated r-chromatin. The OH scavengers were found to give strong protection in the presence of oxygen as anticipated from previous results obtained with this system. By contrast, only a modest protection was observed under 100% N2 or 100% N2O, and the level of protection was different for each scavenger. The data suggest that secondary radicals may inactivate r-chromatin under anoxia. In the presence of oxygen, the secondary radicals react with O2 to form organic peroxy radicals (or O2-) which seem to be less reactive. Since the protective effect of the OH scavengers varies with the gassing conditions, the dose modifying effects of O2 and N2O relative to N2 depend on the identity and concentration of OH scavenger. The implications for radiation-chemical studies on DNA and living cells are discussed.

Inactivation of DNA-mediated transformation of hamster cells by gamma-rays and deoxyribonuclease I.

DNA damage has been induced in the mammalian expression vector pSV2-gpt by irradiation with X-rays or treatment with deoxyribonuclease I (DNAase I) under controlled conditions in vitro. The biological effect of such treatment was assessed by stable gene expression in Chinese hamster ovary (CHO) cells using DNA-mediated gene transfer. Induction of DNA double-strand breaks (dsbs), resulting from the interaction of independently-induced single-strand breaks (ssbs) under the present conditions, was measured by agarose gel electrophoresis of the treated vector. The correlation between radiation-induced gene inactivation and dsb induction mediated by OH radicals suggests that a dsb in the gene is a major inactivating lesion in this system. Individual radiation-induced ssbs and nucleotide damage are produced much more frequently than dsbs under these conditions, but the majority of these lesions do not appear to inactivate the gpt gene. DNAase I treatment, giving only simple 5' P + 3' OH breaks in the vector DNA, gave a correlation of approximately 1.5 dsb in the gpt gene per inactivating event, confirming little repair of dsbs in this system. Inactivation of the gpt gene without appreciable formation of dsbs was found, however, when the vector was irradiated at high dose rate in the presence of the OH-radical scavenger KBr. The nature of non-break damage causing inactivation requires further study.

Single-strand breaks can lead to complex configurations of plasmid DNA in vitro.

DNA species which migrate extremely slowly in agarose gel electrophoresis may be formed from plasmid DNA containing radiation-induced single-strand breaks (ssbs). Postirradiation heat treatment in low ionic strength buffer and subsequent incubation with Mg2+ strongly enhanced the formation of these species. Electron micrographs taken after such treatment show numerous complex configurations containing DNA material from several plasmid molecules. Less extreme formation of slowly migrating DNA occurred without postirradiation heat treatment or Mg2+ incubation when the DNA was co-precipitated with calcium phosphate in a physiologically balanced buffer and incubated under conditions used for DNA-mediated gene transfer. The data suggest that homologous pairing between single-stranded regions formed in relation to ssbs may contribute to cohesion between different molecules. The significance of the cohesion process for gene transfer experiments and cellular radiation effects is discussed.