Lora M. Green

Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation. I. Initial Characterization of DNA Damage, Micronucleus Formation, Apoptosis, Cell Survival, and Cell Cycle Phase Redistribution

Abstract Green, L. M., Murray, D. K., Tran, D. T., Bant, A. M., Kazarians, G., Moyers, M. F. and Nelson, G. A. Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation. I. Initial Characterization of DNA Damage, Micronucleus Formation, Apoptosis, Cell Survival, and Cell Cycle Phase Redistribution. The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to γ rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3′ ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable γ-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by γ rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after γ irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the α components of cell survival curves (0.05 Gy−1 for γ rays, 0.12 Gy−1 for protons), which suggests that the higher level of survival of γ-irradiated cells could be attributed to the persistence of nonlethally irradiated thyrocytes and/or the capacity to repair damage more effectively than cells exposed to equal physical doses of protons. The final assessment in this study was radiation-induced cell cycle phase redistribution. Gamma rays and protons produced a similar dose-dependent redistribution toward a predominantly G2-phase population. From our cumulative results, it seems likely that a majority of the proton-irradiated cells would not continue to divide. In conclusion, these findings suggest that there are quantitative and qualitative differences in the biological effects of proton beams and γ rays. These differences could be due to structured energy deposition from the tracks of primary protons and the associated high-LET secondary particles produced in the targets. The results suggest that a simple dose-equivalent approach to dosimetry may be inadequate to compare the biological responses of cells to photons and protons.

Abnormalities of Serum Antielastin Antibodies in Connective Tissue Diseases

Background Antibodies (Abs) to α-elastin (elastin breakdown product) and tropoelastin (elastin precursor) are found in the serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs, peptides, and elastin metabolism were altered in autoimmune diseases by detecting a changing ratio of serum anti-α:tropoelastin Ab levels. Methods Serum from patients with a variety of connective tissue diseases, including 28 with systemic lupus erythematosus (SLE), 24 with scleroderma, 18 with rheumatoid arthritis (RA), 10 with polymyositis, and 39 with vasculitis, was compared with serum from 19 age-matched healthy subjects for levels of antitropoelastin and anti-α-elastin Abs. Results We found an increase in IgG anti-α-elastin and a decrease in antitropoelastin Abs in the sera of patients with scleroderma (p < .02 and .00005) and SLE (p < .006 and .011). There was also a marked increase in anti-α-elastin Abs in patients with polyarteritis nodosa (p < .0005) and decreases in antitropoelastin Abs in patients with RA (p < .05), polymyositis (p < .01), and a variety of other vasculidities (p < .0003). Conclusions Abnormal variations in elastin metabolism may be detected in several connective tissue diseases by measuring ratios of α- and tropoelastin IgG Abs as markers of elastin degradation and synthesis.

Changes in the activation and reconstitution of lymphocytes resulting from total-body irradiation correlate with slowed tumor growth

Alterations in cytokine secretion, activation marker expression, and immune cell concentrations were investigated at sequential time points following delivery of total-body irradiation (TBI) to C57BL/6 mice (n = 64) in the Lewis lung tumor model. Significantly slower tumor growth was observed when a 3-Gy dose of TBI was administered 2 h prior to tumor implantation (p < 0.05). The antitumor effect was correlated with an increased CD4:CD8 T cell ratio and heightened leukocyte blastogenesis. TBI was also found to induce an expansion of natural killer (NK) cells in the blood and spleen of tumor-bearing animals 10 days after irradiation (2.8 × 106 NK cells/spleen in test mice compared to 8.9 × 105 NK cells/spleen in normal control animals). However, no significant differences were found in NK cell levels within the tumor tissue. Enhanced production of interleukin (IL)-12 and IL-18 from spleen supernatants was consistent with an augmentation of the NK cell response. Significant reductions in transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor, both of which are associated with immune suppression, were also noted. Furthermore, TBI induced changes in expression of CD25 and CD71 activation markers, suggesting that radiation may alter tumor surveillance. Taken together, the relative percentages and activation status of immune cell compartments support the conclusion that these TBI-induced changes function to slow tumor progression.

Alterations in Glutamate Uptake in NT2-Derived Neurons and Astrocytes after Exposure to Gamma Radiation

Abstract Sanchez, M. C., Benitez, A., Ortloff, L. and Green, L. M. Alterations in Glutamate Uptake in NT2-Derived Neurons and Astrocytes after Exposure to Gamma Radiation. Radiat. Res. 171, 41–52 (2009). Currently, the cellular and molecular mechanisms that underlie radiation-induced damage in the CNS are unclear. The present study began investigations of the underlying mechanism(s) for radiation-induced neurotoxicity by characterizing glutamate transport expression and function in neurons and astrocytes after exposure to γ rays. NTera2-derived neurons and astrocytes, isolated as pure cultures, were exposed to doses of 10 cGy, 50 cGy and 2 Gy γ rays, and transporter expression and function were assessed 3 h, 2 days and 7 days after exposure. In neurons, at 7 days after exposure, a significant increase was detected in EAAT3 after 50 cGy (P < 0.05) and a dose-dependent increase in GLT-1 expression was seen between doses of 10 and 50 cGy (P < 0.05). Functional assays of glutamate uptake revealed that neurons and astrocytes respond in a reciprocal manner after irradiation. Neurons responded to radiation exposure by increased glutamate uptake, an effect still evident at our last time (7 days) after exposure (P < 0.05). The astrocyte response to γ radiation was an initial decrease in uptake followed by recovery to baseline levels at 2 days after exposure (P < 0.05). The observations made in this study demonstrate that neurons and astrocytes, while part of the same multifunctional unit, have distinct functional and reciprocal responses. The response in neurons appears to indicate a protracted response with potential long-term effects after irradiation.

Evaluation of Polysaccharopeptide Effects against C6 Glioma in Combination with Radiation

Long-term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. The major goal of this study was to determine whether polysaccharopeptide (PSP), a crude polysaccharide peptide extract derived from Coriolus versicolor, a fungus, could enhance the effects of radiation against glioma cells in culture and in xenografted tumors in vivo. PSP significantly augmented radiation-induced damage to C6 rat glioma cells in vitro. Nude mice injected subcutaneously with the C6 cells were treated with PSP (injected intraperitoneally at 2 mg/injection) and radiation (2 Gy/fraction, 8 Gy in total) using three different time-dose protocols. Tumor volumes were consistently smaller in all treated groups compared to the non-treated tumor-bearing controls except in one group which received PSP prior to tumor implantation. The administration of radiation alone resulted in the slowest tumor progression, whereas PSP alone had no effect. Furthermore, PSP in combination with radiation treatment did not increase radiation efficacy. Natural killer cell, lymphocyte and granulocyte counts in blood and spleen were significantly higher in PSP-treated animals, demonstrating that PSP has protective effects on immunological function. Collectively, these results warrant further investigation to determine if PSP can be effectively utilized to upregulate immune responsiveness in case of neoplasia and other diseases in which immunosuppression is a prominent feature.

Effects of Protons and HZE Particles on Glutamate Transport in Astrocytes, Neurons and Mixed Cultures

Abstract Radiation-induced neurotoxicity is a well-characterized phenomenon. However, the underlying mechanism of this toxicity is poorly understood. In the central nervous system (CNS), excitotoxic mechanisms are implicated in many neurodegenerative disease processes. Pivotal to the excitotoxic pathway is dysfunction of glutamate signaling. We reported previously that exposure to low-LET γ radiation results in altered glutamate transport in neurons and astrocytes. In the present study, we sought to investigate the effects of various particle radiations of differing LET on glutamate transport as a measure of the neurochemical vulnerability of the CNS. NTera2-derived neurons and astrocytes isolated as pure and mixed cultures were exposed to doses of 10 cGy, 50 cGy or 2 Gy of 250 MeV protons, 290 MeV/nucleon carbon ions, or 1000 MeV/nucleon iron ions. Transporter function was assessed at 3 h, 2 days and 7days after exposure. Functional assessment of glutamate transport revealed that neurons and astrocytes respond in a reciprocal manner after exposure to particle radiation. Uptake activity in neurons increased after particle irradiation. This effect was evident as late as our last time (7 days) after exposure (P < 0.05). In astrocytes, transporter activity decreased after exposure. The decrease in uptake observed in astrocytes was evident 7 days after exposure to carbon and iron ions. Uptake in mixed cultures after exposure to all three forms of radiation revealed a muted interactive response suggestive of the individual responses of each cellular phenotype acting in opposition.

Response of thyroid follicular cells to gamma irradiation compared to proton irradiation: II. The role of connexin 32

Abstract Green, L. M., Tran, D. T., Murray, D. K., Rightnar, S. S., Todd, S. and Nelson, G. A. Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation: II. The Role of Connexin 32. Radiat. Res. 158, 475–485 (2002). The objective of this study was to determine whether connexin 32-type gap junctions contribute to the “contact effect” in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after γ irradiation revealed that early-passage follicular cultures had significantly lower values of α (0.04 Gy−1) than later-passage cultures (0.11 Gy−1) (P < 0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher α values, with early-passage cultures (0.10 Gy−1) nearly equivalent to untreated late-passage cultures (0.11 Gy−1) (P > 0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in γ-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had α values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy−1), did not express connexin 32 (0.09 Gy−1), or were down-regulated (early-passage plus heptanol, 0.09 Gy−1; late-passage plus heptanol, 0.12 Gy−1) (P > 0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower α values from the γ-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the β components of the survival curves from both γ rays and proton beams were similar (average 0.022 ± 0.008 Gy–2, P > 0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.

Abnormalities of Serum Anti-Elastin Antibodies in Patients with Polymyalgia Rheumatica

Background: Antibodies [Abs] to alpha-elastin [elastin breakdown product] and tropoelastin [elastin precursor] are found in serum of all human subjects and correlate with their respective serum peptide levels. Serum elastin peptide levels vary with age and some disease states. Vascular damage is thought to be a possible mechanism in the pathogenesis of polymyalgia rheumatica [PMR] and in the closely related condition of giant cell arteritis. Damage to elastin is a characteristic of giant cell arteritis. This study was undertaken to determine if the levels of serum Abs against elastin were altered in patients with PMR by measuring the ratio of serum anti-alpha elastin to anti-tropoelastin Abs compared to age matched controls. Methods: Sera from 37 elderly patients with PMR were compared with sera from 45 age-matched, otherwise healthy osteoarthritis subjects using an enzyme-linked immunosorbent assay that measured levels of anti-alpha and anti-tropoelastin Abs. Results: We found a decrease in anti-tropoelastin and an increase in anti-alpha-elastin immuno-globulin G Abs and in the sera of patients with PMR that were significantly different than the control levels [P < 0.008 [anti-tropoelastin] and 0.005 [anti-alpha-elastin]]. The ratio of anti-tropoelastin [synthesis] to anti-alpha-elastin [degradation] was 1.98 [PMR] versus 3.40 [control] [P < 0.001]. Conclusions: Variations in elastin metabolism were detected in PMR by the ratio of anti-tropoelastin to anti-alpha-elastin immunoglobulin G Abs that represents elastin synthesis and degradation, respectively. This study suggests that there was a decrease in elastin production as well as an increase in elastin destruction in patients with PMR that may be reflecting disease pathology.

IL-2 Gene and Antisense TGF-β1 Strategies Counteract HSV-2 Transformed Tumor Progression

The H238 tumor cells are Herpes simplex virus type 2-transformed BALB/c mouse fibroblasts that constitutively express transforming growth factor (TGF-β1). TGF-β can diminish immune capacity, whereas interleukin 2 (IL-2) is stimulatory to the immune system and can counteract the negative effects of TGF-β1. The H238-BALB/c system provides a syngeneic model to evaluate new strategies with the potential to ameliorate tumor-induced immune depression. Plasmids expressing either antisense TGF-β1 or murine Il-2 were constructed and stably transformed cells generated (masH238 and H238-IL2, respectively). In vitro measurements (ELISA and RT-PCR) demonstrated a >70% decrease in TGF-β1 secretion by the masH238 tumor cells, and significant levels of IL-2 production by the H238-IL2 transfected cells when compared to wild type and control plasmid-transfected H238 cells. BALB/c mice injected subcutaneously with the masH238 cells developed significantly smaller tumors than the controls. Mice injected with H238-IL-2 cells developed tumors that failed to progress relative to control tumor growth. The differences in tumor growth in the mice were associated with enhanced immune reactivity and an increased response to T lymphocyte mitogens. Significant differences were also noted in lymphocyte populations and expression of CD25 and CD71 activation markers in the blood and spleens of mice receiving transfected tumor cells. Collectively, the data demonstrate that strategies employing antisense TGF-β1 and IL-2 expression by transfected tumor cells can counteract the progression of a TGF-β1-secreting tumor and enhance immune function involving modulation of T lymphocyte populations.

Abnormally Low Antibody Markers of Elastin Synthesis in Patients with Fibromyalgia Syndrome

Background: Antibodies [Abs] to tropoelastin [elastin precursor] and alpha-elastin [elastin breakdown product] are found in serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs reflective of elastin metabolism were altered in patients with fibromyalgia syndrome [FMS] by determining the ratio of serum anti-tropoelastin to anti-alpha-elastin Ab levels. Method: Serum samples from 20 female patients with FMS were compared with samples from 19 age-matched healthy normal control subjects for the levels of anti-elastin Abs directed against tropoelastin and alpha elastin using enzyme-linked immunosorbent assays. Results: Anti-tropoelastin Abs in the sera of patients with FMS were decreased relative to the levels measured in healthy subjects [P < 0.0002]. The ratios of anti-tropoelastin to anti-alpha-elastin Abs were 1.98 versus 1.01 for control and FMS subjects, respectively. This suggested a decrease in elastin production in patients with FMS. There were no differences in the average values of anti-alpha-elastin Abs in the FMS patients sera compared to the average value of the control samples, suggesting there was no differences in elastin destruction. Conclusions: Variations in elastin metabolism were detected in sera of patients with FMS. This suggests an altered elastin metabolism in terms of reduced elastin production in patients with FMS. The relationship between elastin metabolism and disease pathology is currently being explored.