Alpha Particle-Emitting Radiopharmaceuticals as Cancer Therapy: Biological Basis, Current Status, and Future Outlook for Therapeutics Discovery.

Critical advances in radionuclide therapy have led to encouraging new options for cancer treatment through the pairing of clinically useful radiation-emitting radionuclides and innovative pharmaceutical discovery. Of the various subatomic particles used in therapeutic radiopharmaceuticals, alpha (α) particles show great promise owing to their relatively large size, delivered energy, finite pathlength, and resulting ionization density. This review discusses the therapeutic benefits of α-emitting radiopharmaceuticals and their pairing with appropriate diagnostics, resulting in innovative "theranostic" platforms. Herein, the current landscape of α particle-emitting radionuclides is described with an emphasis on their use in theranostic development for cancer treatment. Commonly studied radionuclides are introduced and recent efforts towards their production for research and clinical use are described. The growing popularity of these radionuclides is explained through summarizing the biological effects of α radiation on cancer cells, which include DNA damage, activation of discrete cell death programs, and downstream immune responses. Examples of efficient α-theranostic design are described with an emphasis on strategies that lead to cellular internalization and the targeting of proteins involved in therapeutic resistance. Historical barriers to the clinical deployment of α-theranostic radiopharmaceuticals are also discussed. Recent progress towards addressing these challenges is presented along with examples of incorporating α-particle therapy in pharmaceutical platforms that can be easily converted into diagnostic counterparts.

Targeted Inhibition of DNA-PKcs, ATM, ATR, PARP, and Rad51 Modulate Response to X Rays and Protons.

Small molecule inhibitors are currently in preclinical and clinical development for the treatment of selected cancers, particularly those with existing genetic alterations in DNA repair and DNA damage response (DDR) pathways. Keen interest has also been expressed in combining such agents with other targeted antitumor strategies such as radiotherapy. Radiotherapy exerts its cytotoxic effects primarily through DNA damage-induced cell death; therefore, inhibiting DNA repair and the DDR should lead to additive and/or synergistic radiosensitizing effects. In this study we screened the response to X-ray or proton radiation in cell lines treated with DDR inhibitors (DDRis) targeting ATM, ATR, DNA-PKcs, Rad51, and PARP, with survival metrics established using clonogenic assays. We observed that DDRis generate significant radiosensitization in cancer and primary cells derived from normal tissue. Existing genetic defects in cancer cells appear to be an important consideration when determining the optimal inhibitor to use for synergistic combination with radiation. We also show that while greater radiosensitization can be achieved with protons (9.9 keV/µm) combined with DDRis, the relative biological effectiveness is unchanged or in some cases reduced. Our results indicate that while targeting the DDR can significantly radiosensitize cancer cells to such combinations, normal cells may also be equally or more severely affected, depending on the DDRi used. These data highlight the importance of identifying genetic defects as predictive biomarkers of response for combination treatment.

An empirical model of proton RBE based on the linear correlation between x-ray and proton radiosensitivity.

BACKGROUND: Proton relative biological effectiveness (RBE) is known to depend on physical factors of the proton beam, such as its linear energy transfer (LET), as well as on cell-line specific biological factors, such as their ability to repair DNA damage. However, in a clinical setting, proton RBE is still considered to have a fixed value of 1.1 despite the existence of several empirical models that can predict proton RBE based on how a cell's survival curve (linear-quadratic model [LQM]) parameters α and ß vary with the LET of the proton beam. Part of the hesitation to incorporate variable RBE models in the clinic is due to the great noise in the biological datasets on which these models are trained, often making it unclear which model, if any, provides sufficiently accurate RBE predictions to warrant a departure from RBE = 1.1. PURPOSE: Here, we introduce a novel model of proton RBE based on how a cell's intrinsic radiosensitivity varies with LET, rather than its LQM parameters. METHODS AND MATERIALS: We performed clonogenic cell survival assays for eight cell lines exposed to 6 MV x-rays and 1.2, 2.6, or 9.9 keV/µm protons, and combined our measurements with published survival data (n = 397 total cell line/LET combinations). We characterized how radiosensitivity metrics of the form DSF% , (the dose required to achieve survival fraction [SF], e.g., D10% ) varied with proton LET, and calculated the Bayesian information criteria associated with different LET-dependent functions to determine which functions best described the underlying trends. This allowed us to construct a six-parameter model that predicts cells' proton survival curves based on the LET dependence of their radiosensitivity, rather than the LET dependence of the LQM parameters themselves. We compared the accuracy of our model to previously established empirical proton RBE models, and implemented our model within a clinical treatment plan evaluation workflow to demonstrate its feasibility in a clinical setting. RESULTS: Our analyses of the trends in the data show that DSF% is linearly correlated between x-rays and protons, regardless of the choice of the survival level (e.g., D10% , D37% , or D50% are similarly correlated), and that the slope and intercept of these correlations vary with proton LET. The model we constructed based on these trends predicts proton RBE within 15%-30% at the 68.3% confidence level and offers a more accurate general description of the experimental data than previously published empirical models. In the context of a clinical treatment plan, our model generally predicted higher RBE-weighted doses than the other empirical models, with RBE-weighted doses in the distal portion of the field being up to 50.7% higher than the planned RBE-weighted doses (RBE = 1.1) to the tumor. CONCLUSIONS: We established a new empirical proton RBE model that is more accurate than previous empirical models, and that predicts much higher RBE values in the distal edge of clinical proton beams.

Effect of boron compounds on the biological effectiveness of proton therapy.

PURPOSE: We assessed whether adding sodium borocaptate (BSH) or 4-borono-l-phenylalanine (BPA) to cells irradiated with proton beams influenced the biological effectiveness of those beams against prostate cancer cells to investigate if the alpha particles generated through proton-boron nuclear reactions would be sufficient to enhance the biological effectiveness of the proton beams. METHODS: We measured clonogenic survival in DU145 cells treated with 80.4-ppm BSH or 86.9-ppm BPA, or their respective vehicles, after irradiation with 6-MV X-rays, 1.2-keV/µm (low linear energy transfer [LET]) protons, or 9.9-keV/µm (high-LET) protons. We also measured γH2AX and 53BP1 foci in treated cells at 1 and 24 h after irradiation with the same conditions. RESULTS: We found that BSH radiosensitized DU145 cells across all radiation types. However, no difference was found in relative radiosensitization, characterized by the sensitization enhancement ratio or the relative biological effectiveness, for vehicle- versus BSH-treated cells. No differences were found in numbers of γH2AX or 53BP1 foci or γH2AX/53BP1 colocalized foci for vehicle- versus BSH-treated cells across radiation types. BPA did not radiosensitize DU145 cells nor induced any significant differences when comparing vehicle- versus BPA-treated cells for clonogenic cell survival or γH2AX and 53BP1 foci or γH2AX/53BP1 colocalized foci. CONCLUSIONS: Treatment with 11 B, at concentrations of 80.4 ppm from BSH or 86.9 ppm from BPA, had no effect on the biological effectiveness of proton beams in DU145 prostate cancer cells. Our results agree with published theoretical calculations indicating that the contribution of alpha particles from such reactions to the total absorbed dose and biological effectiveness is negligible. We also found that BSH radiosensitized DU145 cells to X-rays, low-LET protons, and high-LET protons but that the radiosensitization was not related to DNA damage.

Outcomes After Breast Radiation Therapy in a Diverse Patient Cohort With a Germline BRCA1/2 Mutation.

PURPOSE: BRCA1/2 pathogenic variant (PV) mutations confer radiation sensitivity preclinically, but there are limited data regarding breast cancer outcomes after radiation therapy (RT) among patients with documented BRCA1/2 PV mutations versus no PV mutations. METHODS AND MATERIALS: This retrospective cohort study included women with clinical stage I-III breast cancer who received definitive surgery and RT and underwent BRCA1/2 genetic evaluation at the The University of Texas MD Anderson Cancer Center. Rates of locoregional recurrence (LRR), disease-specific death (DSD), toxicities, and second cancers were compared by BRCA1/2 PV status. RESULTS: Of the 2213 women who underwent BRCA1/2 testing, 63% self-reported their race as White, 13.6% as Black/African American, 17.6% as Hispanic, and 5.8% as Asian/American Indian/Alaska Native; 124 had BRCA1 and 100 had BRCA2 mutations; and 1394 (63%) received regional nodal RT. The median follow-up time for all patients was 7.4 years (95% confidence interval [CI], 7.1-7.7 years). No differences were found between the groups with and without BRCA1/2 PV mutations in 10-year cumulative incidences of LRR (with mutations: 11.6% [95% CI, 7.0%-17.6%]; without mutations: 6.6% [95% CI, 5.3%-8.0%]; P = .466) and DSD (with mutations: 12.3% [95% CI, 8.0%-17.7%]; without mutations: 13.8% [95% CI, 12.0%-15.8%]; P = .716). On multivariable analysis, BRCA1/2 status was not associated with LRR or DSD, but Black/African American patients (P = .036) and Asians/American Indians/Alaska Native patients (P = .002) were at higher risk of LRR compared with White patients, and Black/African American patients were at higher risk of DSD versus White patients (P = .004). No in-field, nonbreast second cancers were observed in the BRCA1/2 PV group. Rates of acute and late grade ≥3 radiation-related toxicity in the BCRA1/2 PV group were 5.4% (n = 12) and 0.4% (n = 1), respectively. CONCLUSIONS: Oncologic outcomes in a diverse cohort of patients with breast cancer who had a germline BRCA1/2 PV mutation and were treated with RT were similar to those of patients with no mutation, supporting the use of RT according to standard indications in patients with a germline BRCA1/2 PV mutation.

Breast Radiation Therapy-Related Treatment Outcomes in Patients With or Without Germline Mutations on Multigene Panel Testing.

PURPOSE: Multigene panel testing has increased the detection of germline mutations in patients with breast cancer. The implications of using radiation therapy (RT) to treat patients with pathogenic variant (PV) mutations are not well understood and have been studied mostly in women with only BRCA1 or BRCA2 PVs. We analyzed oncologic outcomes and toxicity after adjuvant RT in a contemporary, diverse cohort of patients with breast cancer who underwent genetic panel testing. METHODS AND MATERIALS: We retrospectively reviewed the records of 286 women with clinical stage I-III breast cancer diagnosed from 1995 to 2017 who underwent surgery, breast or chest wall RT with or without regional nodal irradiation, multigene panel testing, and evaluation at a large cancer center's genetic screening program. We evaluated rates of overall survival, locoregional recurrence, disease-specific death, and radiation-related toxicities in 3 groups: BRCA1/2 PV carriers, non-BRCA1/2 PV carriers, and patients without PV mutations. RESULTS: PVs were detected in 25.2% of the cohort (12.6% BRCA1/2 and 12.6% non-BRCA1/2). The most commonly detected non-BRCA1/2 mutated genes were ATM, CHEK2, PALB2, CDH1, TP53, and PTEN. The median follow-up time for the entire cohort was 4.4 years (95% confidence interval, 3.8-4.9 years). No differences were found in overall survival, locoregional recurrence, or disease-specific death between groups (P > .1 for all). Acute and late toxicities were comparable across groups. CONCLUSION: Oncologic and toxicity outcomes after RT in women with PV germline mutations detected by multigene pane testing are similar to those in patients without detectable mutations, supporting the use of adjuvant RT as a standard of care when indicated.

Cell lines of the same anatomic site and histologic type show large variability in intrinsic radiosensitivity and relative biological effectiveness to protons and carbon ions.

PURPOSE: To show that intrinsic radiosensitivity varies greatly for protons and carbon (C) ions in addition to photons, and that DNA repair capacity remains important in governing this variability. METHODS: We measured or obtained from the literature clonogenic survival data for a number of human cancer cell lines exposed to photons, protons (9.9 keV/µm), and C-ions (13.3-77.1 keV/µm). We characterized their intrinsic radiosensitivity by the dose for 10% or 50% survival (D10% or D50% ), and quantified the variability at each radiation quality by the coefficient of variation (COV) in D10% and D50% . We also treated cells with DNA repair inhibitors prior to irradiation to assess how DNA repair capacity affects their variability. RESULTS: We found no statistically significant differences in the COVs of D10% or D50% between any of the radiation qualities investigated. The same was true regardless of whether the cells were treated with DNA repair inhibitors, or whether they were stratified into histologic subsets. Even within histologic subsets, we found remarkable differences in radiosensitivity for high LET C-ions that were often greater than the variations in RBE, with brain cancer cells varying in D10% (D50% ) up to 100% (131%) for 77.1 keV/µm C-ions, and non-small cell lung cancer and pancreatic cancer cell lines varying up to 55% (76%) and 51% (78%), respectively, for 60.5 keV/µm C-ions. The cell lines with modulated DNA repair capacity had greater variability in intrinsic radiosensitivity across all radiation qualities. CONCLUSIONS: Even for cell lines of the same histologic type, there are remarkable variations in intrinsic radiosensitivity, and these variations do not differ significantly between photon, proton or C-ion radiation. The importance of DNA repair capacity in governing the variability in intrinsic radiosensitivity is not significantly diminished for higher LET radiation.

Radiation-Induced Senescence Bystander Effect: The Role of Exosomes.

Ionizing Radiation (IR), especially at high doses, induces cellular senescence in exposed cultures. IR also induces "bystander effects" through signals released from irradiated cells, and these effects include many of the same outcomes observed following direct exposure. Here, we investigate if radiation can cause senescence through a bystander mechanism. Control cultures were exposed directly to 0, 0.1, 2, and 10 Gy. Unirradiated cells were treated with medium from irradiated cultures or with exosomes extracted from irradiated medium. The level of senescence was determined post-treatment (24 h, 15 days, 30 days, and 45 days) by ß-galactosidase staining. Media from cultures exposed to all four doses, and exosomes from these cultures, induced significant senescence in recipient cultures. Senescence levels were initially low at the earliest timepoint, and peaked at 15 days, and then decreased with further passaging. These results demonstrate that senescence is inducible through a bystander mechanism. As with other bystander effects, bystander senescence was induced by a low radiation dose. However, unlike other bystander effects, cultures recovered from bystander senescence after repeated passaging. Bystander senescence may be a potentially significant effect of exposure to IR, and may have both beneficial and harmful effects in the context of radiotherapy.

Isolation of time-dependent DNA damage induced by energetic carbon ions and their fragments using fluorescent nuclear track detectors.

PURPOSE: High energetic carbon (C-) ion beams undergo nuclear interactions with tissue, producing secondary nuclear fragments. Thus, at depth, C-ion beams are composed of a mixture of different particles with different linear energy transfer (LET) values. We developed a technique to enable isolation of DNA damage response (DDR) in mixed radiation fields using beam line microscopy coupled with fluorescence nuclear track detectors (FNTDs). METHODS: We imaged live cells on a coverslip made of FNTDs right after C-ion, proton or photon irradiation using an in-house built confocal microscope placed in the beam path. We used the FNTD to link track traversals with DNA damage and separated DNA damage induced by primary particles from fragments. RESULTS: We were able to spatially link physical parameters of radiation tracks to DDR in live cells to investigate spatiotemporal DDR in multi-ion radiation fields in real time, which was previously not possible. We demonstrated that the response of lesions produced by the high-LET primary particles associates most strongly with cell death in a multi-LET radiation field, and that this association is not seen when analyzing radiation induced foci in aggregate without primary/fragment classification. CONCLUSIONS: We report a new method that uses confocal microscopy in combination with FNTDs to provide submicrometer spatial-resolution measurements of radiation tracks in live cells. Our method facilitates expansion of the radiation-induced DDR research because it can be used in any particle beam line including particle therapy beam lines. CATEGORY: Biological Physics and Response Prediction.

Nonhomologous End Joining Is More Important Than Proton Linear Energy Transfer in Dictating Cell Death.

PURPOSE: This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. METHODS AND MATERIALS: We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs-/-), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. RESULTS: Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. CONCLUSIONS: BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.

Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells.

PURPOSE: To investigate the effects of ionizing radiation on telomere length and telomerase activity in human lens epithelial cells. There are studies suggesting evidence of telomere length in association with opacity of the lens; however, these studies have been conducted on Canine Lens cells. Our study was designed to understand further the effects of different doses of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells from three Donors. MATERIALS AND METHODS: For this study, embryonic human lens epithelial (HLE) cells from three donors, obtained commercially were cultured. Telomere length and telomerase activity were measured after each passage until cells stopped growing in culture. This was repeated on irradiated (0.001 Gy, 0.01 Gy, 0.02 Gy, 0.1 Gy, 1 Gy and 2 Gy) cells. DNA damage response using the H2AX and telomere dysfunction foci assays were also examined at 30 mins, 24 hours, 48 hours and 72 hours postirradiation. RESULTS AND CONCLUSION: We have demonstrated genetic changes in telomere length and oxidative stress, which may be relevant to cataractogenesis. Our study shows that in control cells telomere length increases as passage increases. We have also demonstrated that telomere length increases at higher doses of 1.0 Gy and 2.0 Gy. However, telomerase activity decreases dose dependently and as passages increase. These results are not conclusive and further studies ex vivo measuring lens opacity and telomere length in the model would be beneficial in a bigger cohort, hence confirming a link between telomere length, cataractogenesis and genetic factors.

The future impacts of non-targeted effects.

Ionizing radiation was traditionally thought to exert its detrimental effects through interaction with sensitive cellular targets, nuclear DNA being of most importance. This theory has since merged with a more recently described radiation response called non-targeted effects (NTE). This review will briefly look at the various types of NTE and the potential implications they may have for radiobiology research and its applications. The most well-known NTE are genomic instability (GI) and bystander effects (BE). Other NTE include abscopal effects, which are similar to bystander effects but are generally based in a clinical environment with immune involvement as the defining feature. Currently, our understanding of NTE is limited to certain signaling pathways/molecules, and as yet there is no theory that describes or can accurately predict the occurrence or outcome of these NTE. There are numerous groups investigating these processes in vitro and in vivo, and thus steady progress is being made. Developing a deeper understanding of NTE has potential impacts for therapy and diagnosis, safer occupational exposures, space flight and our general understanding of radiation biology.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen.

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

Exosome-Mediated Telomere Instability in Human Breast Epithelial Cancer Cells after X Irradiation.

In directly irradiating cells, telomere metabolism is altered and similar effects have been observed in nontargeted cells. Exosomes and their cargo play dominant roles in communicating radiation-induced bystander effects with end points related to DNA damage. Here we report novel evidence that exosomes are also responsible for inducing telomere-related bystander effects. Breast epithelial cancer cells were exposed to either 2 Gy X rays, or exposed to irradiated cell conditioned media (ICCM), or exosomes purified from ICCM. Compared to control cells, telomerase activity decreased in the 2 Gy irradiated cells and both bystander samples after one population doubling. At the first population doubling, telomere length was shorter in the 2 Gy irradiated sample but not in the bystander samples. By 24 population doublings telomerase activity recovered to control levels in all samples; however, the 2 Gy irradiated sample continued to demonstrate short telomeres and both bystander samples acquired shorter telomeres. RNase treatment of exosomes prevented the bystander effects on telomerase and telomere length that were observed at 1 population doubling and 24 population doublings, respectively. Thermal denaturation by boiling eliminated the reduction of telomere length in bystander samples, suggesting that the protein fraction of exosomes also contributes to the telomeric effect. RNase treatment plus boiling abrogated all telomere-related effects in directly irradiated and bystander cell populations. These findings suggest that both proteins and RNAs of exosomes can induce alterations in telomeric metabolism, which can instigate genomic instability in epithelial cancer cells after X-ray irradiation.

Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research.

The lens of the eye has long been considered as a radiosensitive tissue, but recent research has suggested that the radiosensitivity is even greater than previously thought. The 2012 recommendation of the International Commission on Radiological Protection (ICRP) to substantially reduce the annual occupational equivalent dose limit for the ocular lens has now been adopted in the European Union and is under consideration around the rest of the world. However, ICRP clearly states that the recommendations are chiefly based on epidemiological evidence because there are a very small number of studies that provide explicit biological, mechanistic evidence at doses <2Gy. This paper aims to present a review of recently published information on the biological and mechanistic aspects of cataracts induced by exposure to ionizing radiation (IR). The data were compiled by assessing the pertinent literature in several distinct areas which contribute to the understanding of IR induced cataracts, information regarding lens biology and general processes of cataractogenesis. Results from cellular and tissue level studies and animal models, and relevant human studies, were examined. The main focus was the biological effects of low linear energy transfer IR, but dosimetry issues and a number of other confounding factors were also considered. The results of this review clearly highlight a number of gaps in current knowledge. Overall, while there have been a number of recent advances in understanding, it remains unknown exactly how IR exposure contributes to opacification. A fuller understanding of how exposure to relatively low doses of IR promotes induction and/or progression of IR-induced cataracts will have important implications for prevention and treatment of this disease, as well as for the field of radiation protection.

The non-targeted effects of radiation are perpetuated by exosomes.

Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE, spread these effects to naïve cells, and perpetuate GI in the affected cells.

Regulation of human cone cyclic nucleotide-gated channels by endogenous phospholipids and exogenously applied phosphatidylinositol 3,4,5-trisphosphate.

Cyclic nucleotide-gated (CNG) channels are critical components of the vertebrate visual transduction cascade involved in converting light-induced changes in intracellular cGMP concentrations into electrical signals that can be interpreted by the brain as visual information. To characterize regulatory mechanisms capable of altering the apparent ligand affinity of cone channels, we have expressed heteromeric (CNGA3 + CNGB3) human cone CNG channels in Xenopus laevis oocytes and characterized the alterations in channel activity that occur after patch excision using patch-clamp recording in the inside-out configuration. We found that cone channels exhibit spontaneous changes in current at subsaturating cGMP concentrations; these changes are enhanced by application of ATP and seem to reflect alterations in channel gating. Similar to rod CNG channels, lavendustin A prevented this regulation, suggesting the involvement of a tyrosine phosphorylation event. However, the tyrosine residue in CNGB3 (Tyr545) that is equivalent to the critical tyrosine residues in rod and olfactory CNG channel subunits does not participate in cone channel regulation. Furthermore, the changes in ligand sensitivity of CNGA3 + CNGB3 channels were prevented by inhibition of phosphatidylinositol 3-kinase (PI3-kinase) using wortmannin or 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which suggests that phospholipid metabolism can regulate the channels. Direct application of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to the intracellular face of excised patches also resulted in down-regulation of channel activity. Thus, phospholipid metabolism and exogenously applied PIP3 can modulate heterologously expressed cone CNG channels.

Disease-associated mutations in CNGB3 produce gain of function alterations in cone cyclic nucleotide-gated channels.

PURPOSE: To characterize the functional consequences of disease-associated mutations in the CNGB3 (B3) subunit of human cone photoreceptor cyclic nucleotide-gated channels in order to gain insight into disease mechanisms. METHODS: Three separate disease-associated mutations were generated in CNGB3: F525N, R403Q, and T383fsX. These mutant subunits were then heterologously expressed in Xenopus oocytes in combination with wild type CNGA3 (A3) subunits, and characterized by patch-clamp recording in the inside-out configuration. RESULTS: Co-expression of A3 and B3F525N, A3 and B3R403Q, or A3 and B3R403Q and B3T383fsX subunits resulted in channels that exhibited an increase in ligand sensitivity without a reduction in current density compared to wild-type heteromeric channels. Each simulated disease state produced channels that exhibited greater sensitivity to block by L-cis-diltiazem than homomeric CNGA3 channels, confirming that the mutant CNGB3 subunits were competent to form functional heteromeric channels. Each combination of subunits displayed an increase in apparent affinity for cGMP relative to wild-type heteromeric channels. However, F525N enhanced cGMP apparent affinity to a significantly greater extent than the other two modeled disease states. CONCLUSIONS: We have examined the gating effects of two previously uncharacterized disease-associated mutations in the CNGB3 subunit and found that in each case, the mutations resulted in a gain of function molecular phenotype. Furthermore, the magnitude of the effect on channel function correlated with the severity of the associated disease. The complete achromatopsia-associated F525N mutation resulted in more pronounced alterations in channel function than the mutation combinations linked to macular degeneration or progressive cone dystrophy.