Post-surgical lymphedema frequently occurs following lymph node dissection. The murine tail is the most commonly used model to study secondary lymphedema. The murine hindlimb model offers a more clinically translatable approach but results in the literature have been inconsistent. The purpose of this study is to optimize the murine hindlimb lymphedema to achieve consistent results and assess the utility of radiation. C57BL/6 mice underwent either 20-Gy irradiation of one hindlimb 7 days prior to surgery (n = 11) or no radiation (n = 9). For all mice, a circumferential skin incision was created at the proximal hindlimb. Lymphatics were identified and disrupted. Popliteal lymph nodes were excised. Paw thickness was measured and near-infrared laser lymphangiography was used to assess lymphatic function. The average paw thickness of the operated hindlimb in irradiated mice on postoperative day (POD) 14 was 3.5 ± 0.3 cm compared to 2.1 ± 0.05 cm on the contralateral limb (p = 0.0001). Lymphangiography on POD-42 showed significantly worse lymphatic function in the operated hindlimb compared to the control hindlimb (p = 0.003). For the non-radiated mice, the paw thickness was 2.5 ± 0.2 cm on POD-42 compared to the contralateral limb (2.1 ± 0.1 cm) (p = 0.0002) but smaller than radiated hindlimb group (3.2 ± 0.1 cm) (p = 0.0002). The nonradiated mice had significantly greater paw thickness than the control limb until POD-56 whereas the radiated mice sustained significant paw thickness to POD-90. Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model with consistent results.

PurposeFOS-like antigen 1 (FRA1), encoded by FOSL1, is an inducible subunit of the AP-1 transcription factor complex and regulates gene expression in response to proliferative and environmental cues. Although FRA1 has been linked to cancer progression, its role in early transformation and radiation responses remains unclear.MethodsCRISPR-engineered human CGL1 cells—a hybrid of HeLa and normal fibroblasts—were used to evaluate the impact of FRA1 overexpression and knockout on neoplastic transformation. Transformation frequency, clonogenic survival, DNA damage recognition and repair, and cell cycle distribution were assessed following irradiation. Transcriptomic profiling was performed under baseline and serum-stimulated conditions.ResultsFRA1 loss markedly increased both spontaneous and radiation-induced transformation frequency, while overexpression suppressed transformation under both conditions. FRA1-deficient cells were sensitized to radiation-induced cell killing, despite intact DNA damage recognition and repair. In contrast, FRA1 overexpression promoted G2/M accumulation post-irradiation, suggesting enhanced checkpoint activation. Transcriptomic profiling revealed that FRA1 remodels AP-1 complex composition and functions as a transcriptional repressor of mitogen- and stress-responsive genes. FRA1-mediated repression was observed across gene networks involved in extracellular matrix remodeling, hypoxia signaling, inflammation, and proliferation, under both baseline and serum-stimulated conditions.ConclusionThese findings establish FRA1 as a key modulator of neoplastic transformation and radiation response, acting primarily through transcriptional repression of pro-tumorigenic signaling pathways.

Natural background ionizing radiation is present on the earth's surface; however, the biological role of this chronic low-dose-rate exposure remains unknown. The Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) project is examining the impacts of sub-natural background radiation exposure through experiments conducted 2 km underground in SNOLAB. The rock overburden combined with experiment-specific shielding provides a background radiation dose rate 30 times lower than on the surface. We hypothesize that natural background radiation is essential for life and maintains genomic stability and that prolonged exposure to sub-background environments will be detrimental to biological systems. To evaluate this, human hybrid CGL1 cells were continuously cultured in SNOLAB and our surface control laboratory for 16 weeks. Cells were assayed every 4 weeks for growth rate, alkaline phosphatase (ALP) activity (a marker of cellular transformation in the CGL1 system), and the expression of genes related to DNA damage and cell cycle regulation. A subset of cells was also exposed to a challenge radiation dose (0.1 to 8 Gy of X rays) and assayed for clonogenic survival and DNA double-strand break induction to examine if prolonged sub-background exposure alters the cellular response to high-dose irradiation. At each 4-week time point, sub-background radiation exposure did not significantly alter cell growth rates, survival, DNA damage, or gene expression. However, cells cultured in SNOLAB showed significantly higher ALP activity, a marker of carcinogenesis in these cells, which increased with longer exposure to the sub-background environment, indicative of neoplastic progression. Overall, these data suggest that sub-background radiation exposure does not impact growth, survival, or DNA damage in CGL1 cells but may lead to increased rates of neoplastic transformation, highlighting a potentially important role for natural background radiation in maintaining normal cellular function and genomic stability.

The aim of this paper is to review the history surrounding the discovery of lethal mutations, later described as delayed reproductive death. Lethal mutations were suggested very early on, to be due to a generalised instability in a cell population and are considered now to be one of the first demonstrations of "radiation-induced genomic instability" which led later to the establishment of the field of "non-targeted effects." The phenomenon was first described by Seymour et al. in 1986 and was confirmed by Trott's group in Europe and by Little and colleagues in the United States before being extended by Mendonca et al. in 1989, who showed conclusively that the distinguishing feature of lethal mutation occurrence was that it happened suddenly after about 9-10 - 10 population doublings in progeny which had survived the original dose of ionizing radiation. However, many authors then suggested that in fact, lethal mutations were implicit in the original experiments by Puck and Marcus in 1956 and were described in the extensive work by Sinclair in 1964, who followed clonal progeny for up to a year after irradiation and described "small colony formation" as a persistent consequence of ionizing radiation exposure. In this paper, we examine the history from 1956 to the present using the period from 1986-1989 - 1989 as an anchor point to reach into the past and to go forward through the evolution of the field of low dose radiobiology where non-targeted effects predominate. (c) 2024 by Radiation Research Society

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2–3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

PDF file - 372K, Supplementary Figure 1 (Fig.S1)- Detection of CD24 transcript in AsPC1 cells, Supplementary Figure 2 (Fig.S2) Detection of Hh target genes in orthotopic pancreatic cancer models, Supplementary Figure 3 (Fig.S3) Expression of SHH and IHH in AsPC1-derived tumors, Supplementary Figure 4 (Fig.S4) Expression of mouse Hh target genes in AsPC1- and MIA PaCa2-derived tumors, Supplementary Figure 5 (Fig.S5) Picture of tumor spheres (100X) from AsPC1-derived pancreatic tumors or lymph node metastases, Supplementary Figure 6 (Fig.S6) Effect of CycT on tumor sphere regeneration, Supplementary Figure 7 (Fig.S7) Effects of combining radiation with Hh signaling inhibition on tumor sphere regeneration of MMC16 cells, Supplementary Figure 8 (Fig.S8) Effects of Hh signaling inhibitor BMS833923 on Hh target gene expression in AsPC1 derived orthotopic model, Supplementary Figure 9 (Fig.S9) Effects of irradiation and Hh signaling inhibitor BMS (BMS833932) on lung metastasis, Supplementary Figure 10 (Fig.S10) Regulation of putative Hh target genes by Hh signaling inhibition in pancreatic cancer

In 2017, a special edition of Radiation Research was published [Oct; Vol. 188 4.2 (https://bioone.org/journals/radiation-research/volume-188/issue-4.2)] which focused on a recently established radiobiology project within SNOLAB, a unique deep-underground research facility. This special edition included original articles, reviews and commentaries relevant to the research goals of this new project which was titled Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR). These research goals were founded in understanding the biological effects of terrestrial and cosmic natural background radiation (NBR). Since 2017, REPAIR has evolved into a sub-NBR radiobiology research program which investigates these effects using multiple model systems and various biological endpoints. This paper summarizes the evolution of the REPAIR project over the first 6-years including its experimental scope and capabilities as well as research accomplishments.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have exhibited great promise in the treatment of tumors with homologous recombination (HR) deficiency, however, PARPi resistance, which ultimately recovers DNA repair and cell progress, has become an enormous clinical challenge. Recently, KP372-1 was identified as a novel potential anticancer agent that targeted the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce extensive reactive oxygen species (ROS) generation that amplified DNA damage, leading to cancer cell death. To overcome PARPi resistance and expand its therapeutic utility, we investigated whether a combination therapy of a sublethal dose of KP372-1 with a nontoxic dose of PARPi rucaparib would synergize and enhance lethality in NQO1 over-expressing cancers. We reported that the combination treatment of KP372-1 and rucaparib induced a transient and dramatic AKT hyperactivation that inhibited DNA repair by regulating FOXO3a/GADD45α pathway, which enhanced PARPi lethality and overcame PARPi resistance. We further found that PARP inhibition blocked KP372-1-induced PARP1 hyperactivation to reverse NAD+/ATP loss that promoted Ca2+-dependent autophagy and apoptosis. Moreover, pretreatment of cells with BAPTA-AM, a cytosolic Ca2+ chelator, dramatically rescued KP372-1- or combination treatment-induced lethality and significantly suppressed PAR formation and γH2AX activation. Finally, we demonstrated that this combination therapy enhanced accumulation of both agents in mouse tumor tissues and synergistically suppressed tumor growth in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1+ solid tumors that may broaden the clinical utility of PARPi.

Background: Unfolded protein responses (UPR) determine cell fate and are recognized as anticancer targets. In a previous research, we reported that cryptotanshinone (CPT) exerted cytotoxic effects toward acute lymphoblastic leukemia cells through mitochondria-mediated apoptosis.Purpose: In the present study, we further investigated the role of UPR in CPT-induced cytotoxicity on acute lymphoblastic leukemia cells by applying tools of pharmacogenomics and bioinformatics.Methods: Gene expression profiling was performed by mRNA microarray hybridization. Potential transcription factor binding motifs were identified in the promoter regions of the deregulated genes by Cistrome software. Molecular docking on eIF-4A and PI3K was performed to investigate the inhibitory activity of CPT on translation initiation.Results: CPT regulated genes related to UPR and eIF2 signaling pathways. The DNA-Damage-Inducible Transcript 3 (DDIT3) gene, which is activated as consequence of UPR malfunction during apoptosis, was induced and validated by in vitro experiments. Transcription factor binding motif analysis of the microarrary-retrieved deregulated genes in the promoter region emphasized the relevance of transcription factors, such as ATF2, ATF4 and XBP1, regulating UPR and cell apoptosis. Molecular docking suggested inhibitory effects of CPT by binding to eIF-4A and PI3K providing evidence for a role of CPT's in the disruption of protein synthesis.Conclusion: CPT triggered UPR and inhibited protein synthesis via eIF-mediated translation initiation, potentially supporting CPT-induced cytotoxic effects toward acute leukemia cells.

This chapter covers a basic understanding of cancer biology, including the role of oncogenes and tumour suppressor genes and their role in controlling the cell cycle. Relevant links are made throughout to applying this knowledge to recent advances in cancer treatment, including immunology and targeted therapies. It also explores metastases and the complex mechanisms involved which enable cancer cells to spread throughout the body. Finally it looks briefly at tumour classification.