Marc S. Mendonca

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects.

Abstract Lewis, D. A., Mayhugh, B. M., Qin, Y., Trott, K. and Mendonca, M. S. Production of Delayed Death and Neoplastic Transformation in CGL1 Cells by Radiation-Induced Bystander Effects. Radiat. Res. 156, 251–258 (2001). Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence α particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 ± 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 × 10–6 in unirradiated controls to 2.3 × 10–5 (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.

DNA damage-induced phosphorylation of the human telomere-associated protein TRF2

Several protein kinases from diverse eukaryotes known to perform important roles in DNA repair have also been shown to play critical roles in telomere maintenance. Here, we report that the human telomere-associated protein TRF2 is rapidly phosphorylated in response to DNA damage. We find that the phosphorylated form of TRF2 is not bound to telomeric DNA, as is the ground form of TRF2, and is rapidly localized to damage sites. Our results suggest that the ataxia-telangiectasia-mutated (ATM) protein kinase signal-transduction pathway is primarily responsible for the DNA damage-induced phosphorylation of TRF2. Unlike DNA damage-induced phosphorylation of other ATM targets, the phosphorylated form of TRF2 is transient, being detected rapidly at DNA damage sites postirradiation, but largely dissipated by 2 hours. In addition, we report that the phosphorylated form of TRF2 is present at telomeres in cell types undergoing telomere-based crisis and a recombination-driven, telomerase-independent, alternative lengthening of telomeres (ALT) pathway, likely as a consequence of a telomere-based DNA damage response. Our results link the induction of TRF2 phosphorylation to the DNA damage-response system, providing an example of direct cross-talk via a signaling pathway between these two major cellular processes essential for genomic stability, telomere maintenance, and DNA repair.

Variations in the RBE for Cell Killing Along the Depth-Dose Profile of a Modulated Proton Therapy Beam

Considerable evidence now exists to show that that the relative biological effectiveness (RBE) changes considerably along the proton depth-dose distribution, with progressively higher RBE values at the distal part of the modulated, or spread out Bragg peak (SOBP) and in the distal dose fall-off (DDF). However, the highly variable nature of the existing studies (with regards to cell lines, and to the physical properties and dosimetry of the various proton beams) precludes any consensus regarding the RBE weighting factor at any position in the depth-dose profile. We have thus conducted a systematic study on the variation in RBE for cell killing for two clinical modulated proton beams at Indiana University and have determined the relationship between the RBE and the dose-averaged linear energy transfer (LETd) of the protons at various positions along the depth-dose profiles. Clonogenic assays were performed on human Hep2 laryngeal cancer cells and V79 cells at various positions along the SOBPs of beams with incident energies of 87 and 200 MeV. There was a marked variation in the radiosensitivity of both cell lines along the SOBP depth-dose profile of the 87 MeV proton beam. Using Hep2 cells, the D0.1 isoeffect dose RBE values (normalized against 60Co) were 1.46 at the middle of SOBP, 2.1 at the distal end of the SOBP and 2.3 in the DDF. For V79 cells, the D0.1 isoeffect RBE for the 87 MEV beam were 1.23 for the proximal end of the SOBP: 1.46 for the distal SOBP and 1.78 for the DDF. Similar D0.1 isoeffect RBE values were found for Hep2 cells irradiated at various positions along the depth-dose profile of the 200 MeV beam. Our experimentally derived RBE values were significantly correlated (P = 0.001) with the mean LETd of the protons at the various depths, which confirmed that proton RBE is highly dependent on LETd. These in vitro data suggest that the RBE of the proton beam at certain depths is greater than 1.1, a value currently used in most treatment planning algorithms. Thus, the potential for increased cell killing and normal tissue damage in the distal regions of the proton SOBP may be greater than originally thought.

Delayed heritable damage and epigenetics in radiation-induced neoplastic transformation of human hybrid cells.

The HeLa x skin fibroblast human hybrid cell system has proven to be an excellent model system for quantitative studies of radiation-induced neoplastic transformation in vitro. A unique aspect of this system is the reexpression of a cell surface protein p75/150 with tumorigenicity. The identification of p75/150 as intestinal alkaline phosphatase (IAP) allowed for the recent development of a more simplified, rapid, and sensitive screening method than the previous p75/150 antibody-based staining procedure. The new method directly detects neoplastically transformed, IAP-expressing cells by staining with the alkaline phosphatase chromogenic substrate, Western Blue (WB). Earlier studies with the antibody-based immunoperoxidase assay indicated that, while no foci with tumor-associated antigen (p75-positive) were evident 15 days after irradiation, the number of foci rose quickly and leveled off between Day 19 and Day 23. This late appearance of the IAP-positive foci suggested that the neoplastic transformation process was not an immediate consequence of radiation damage. The mechanism underlying this observation was unknown. The possibility existed that very small foci and/or foci expressing a low level of IAP were being missed at earlier expression times. The increased sensitivity of the WB staining technique has allowed for the reinvestigation of the kinetics of induction of radiation-induced foci in this system. Experiments were performed where parallel groups of transformation flasks were stained at Days 7, 9, 11, 13, 15, 17, 19, and 21 days after irradiation. The data clearly indicate that the radiation induction of IAP-positive foci is indeed delayed in this system with the vast majority of the foci beginning to appear after Day 9 after irradiation. The delay is not the result of a lack of ability to detect small IAP-positive foci since foci with as few as 15 IAP-positive cells were discernible. We have reported previously that under identical experimental conditions both the establishment of plateau phase and the onset of the expression of lethal mutations also occur after Day 9. We therefore propose that radiation-induced neoplastic transformation of HeLa x skin fibroblast hybrid cells is a consequence of the delayed expression of heritable damage under epigenetic control with a resultant loss of tumor-suppressor function.

A simplified and rapid staining method for the HeLa × skin fibroblast human hybrid cell neoplastic transformation assay

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

Radiation-induced genetic instability in vivo depends on p53 status.

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.

Use of the Leksell Gamma Knife for localized small field lens irradiation in rodents.

For most basic radiobiological research applications involving irradiation of small animals, it is difficult to achieve the same high precision dose distribution realized with human radiotherapy. The precision for irradiations performed with standard radiotherapy equipment is ±2 mm in each dimension, and is adequate for most human treatment applications. For small animals such as rodents, whose organs and tissue structures may be an order of magnitude smaller than those of humans, the corresponding precision required is closer to ±0.2 mm, if comparisons or extrapolations are to be made to human data. The Leksell Gamma Knife is a high precision radiosurgery irradiator, with precision in each dimension not exceeding 0.5 mm, and overall precision of 0.7 mm. It has recently been utilized to treat ocular melanoma and induce targeted lesions in the brains of small animals. This paper describes the dosimetry and a technique for performing irradiation of a single rat eye and lens with the Gamma Knife while allowing the contralateral eye and lens of the same rat to serve as the “control”. The dosimetry was performed with a phantom in vitro utilizing a pinpoint ion chamber and thermoluminescent dosimeters, and verified by Monte Carlo simulations. We found that the contralateral eye received less than 5% of the administered dose for a 15 Gy exposure to the targeted eye. In addition, after 15 Gy irradiation 15 out of 16 animals developed cataracts in the irradiated target eyes, while 0 out of 16 contralateral eyes developed cataracts over a 6-month period of observation. Experiments at 5 and 10 Gy also confirmed the lack of cataractogenesis in the contralateral eye. Our results validate the use of the Gamma Knife for cataract studies in rodents, and confirmed the precision and utility of the instrument as a small animal irradiator for translational radiobiology experiments.

Targeting superoxide dismutase 1 to overcome cisplatin resistance in human ovarian cancer.

PurposeClinical drug resistance to platinum-based chemotherapy is considered a major impediment in the treatment of human ovarian cancer. Multiple pathways associated with drug resistance have been suggested by many previous studies. Over expression of several key proteins involved in DNA repair, drug transport, redox regulation, and apoptosis has been recently reported by our group using a global quantitative proteomic profiling approach. Superoxide dismutase 1 (SOD1) is one of these proteins consistently over-expressed in cisplatin-resistant ovarian cancer cells as compared to their sensitive counterparts, but its precise role in drug resistance is yet to be defined.MethodIn the current study, we examined the role of SOD1 in drug resistance by inhibiting its redox activity in cisplatin-resistant ovarian cancer cells using a small-molecule inhibitor, triethylenetetramine (TETA). The effect of TETA was determined by the cell proliferation assay, clonogenic cell survival assay, and SOD1 activity assay.ResultsThe inhibition of the SOD1 activity enhanced the cisplatin sensitivity in the resistant cells supporting the hypothesis that SOD1 is a key determinant of cisplatin resistance and is an exploitable target to overcome cisplatin drug resistance.ConclusionSOD1 plays an important role in cisplatin resistance and modulation of its activity may overcome this resistance and ultimately lead to improved clinical outcomes.

Modification of pH induced cellular inactivation by irradiation—glial cells☆

Abstract Human cells of glial origin were examined for clonogenic survival after exposure to subnormal pH of the extracellular fluid for 0 to 5 days at 37°C. The surviving fraction decreased at pH values between 6.7 and 6.4 to 10% within 2 days. Cells that survived irradiation demonstrated a decreased sensitivity to an acid extracellular fluid. The radiation response function was established with and without incubation at pH 6.7 for 66 hours. Doses above 4 Gray resulted in a higher survival at pH 6.7 than at pH 7.4, if pH was reduced 15 hours prior to irradiation. Enhanced clonogenic survival after irradiation under conditions of continuous acidity in the extracellular space may be a further explanation for poor local control of malignant tumors in clinical radiation therapy.

DNA damage-induced phosphorylation of TRF2 is required for the fast pathway of DNA double-strand break repair.

ABSTRACT Protein kinases of the phosphatidylinositol 3-kinase-like kinase family, originally known to act in maintaining genomic integrity via DNA repair pathways, have been shown to also function in telomere maintenance. Here we focus on the functional role of DNA damage-induced phosphorylation of the essential mammalian telomeric DNA binding protein TRF2, which coordinates the assembly of the proteinaceous cap to disguise the chromosome end from being recognized as a double-stand break (DSB). Previous results suggested a link between the transient induction of human TRF2 phosphorylation at threonine 188 (T188) by the ataxia telangiectasia mutated protein kinase (ATM) and the DNA damage response. Here, we report evidence that X-ray-induced phosphorylation of TRF2 at T188 plays a role in the fast pathway of DNA DSB repair. These results connect the highly transient induction of human TRF2 phosphorylation to the DNA damage response machinery. Thus, we find that a protein known to function in telomere maintenance, TRF2, also plays a functional role in DNA DSB repair.