Publications by authors named "Jerry W Shay"

282 Publications

Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe.

J Cell Biol 2021 Jun;220(6)

Department of Medicine, Washington University in St. Louis, St. Louis, MO.

It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.
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http://dx.doi.org/10.1083/jcb.202011014DOI Listing
June 2021

Telomeres and replicative cellular aging of the human placenta and chorioamniotic membranes.

Sci Rep 2021 Mar 4;11(1):5115. Epub 2021 Mar 4.

Center of Human Development and Aging (F-464 MSB), New Jersey Medical School, Rutgers, The State University of New Jersey, 185 South Orange Ave, Newark, NJ, 07103, USA.

Recent hypotheses propose that the human placenta and chorioamniotic membranes (CAMs) experience telomere length (TL)-mediated senescence. These hypotheses are based on mean TL (mTL) measurements, but replicative senescence is triggered by short and dysfunctional telomeres, not mTL. We measured short telomeres by a vanguard method, the Telomere shortest length assay, and telomere-dysfunction-induced DNA damage foci (TIF) in placentas and CAMs between 18-week gestation and at full-term. Both the placenta and CAMs showed a buildup of short telomeres and TIFs, but not shortening of mTL from 18-weeks to full-term. In the placenta, TIFs correlated with short telomeres but not mTL. CAMs of preterm birth pregnancies with intra-amniotic infection showed shorter mTL and increased proportions of short telomeres. We conclude that the placenta and probably the CAMs undergo TL-mediated replicative aging. Further research is warranted whether TL-mediated replicative aging plays a role in all preterm births.
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http://dx.doi.org/10.1038/s41598-021-84728-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933277PMC
March 2021

Imaging assay to probe the role of telomere length shortening on telomere-gene interactions in single cells.

Chromosoma 2021 Mar 8;130(1):61-73. Epub 2021 Feb 8.

Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA.

Telomeres are repetitive non-coding nucleotide sequences (TTAGGGn) capping the ends of chromosomes. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Here, we describe an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. We examined two human genes, ISG15 and TERT, for which TPE-OLD has been described before. For both genes, we found less interaction with the telomere on the same chromosome in old cells compared to young cells; and experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.
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http://dx.doi.org/10.1007/s00412-020-00747-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889534PMC
March 2021

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway.

Cancer Cell 2021 Jan 17;39(1):109-121.e5. Epub 2020 Dec 17.

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address:

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.
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http://dx.doi.org/10.1016/j.ccell.2020.11.004DOI Listing
January 2021

Accelerating drug development for neuroblastoma: Summary of the Second Neuroblastoma Drug Development Strategy forum from Innovative Therapies for Children with Cancer and International Society of Paediatric Oncology Europe Neuroblastoma.

Eur J Cancer 2020 09 9;136:52-68. Epub 2020 Jul 9.

Paediatric Drug Development, Children & Young People's Unit, The Royal Marsden NHS Foundation Trust, Sutton, UK; Division of Clinical Studies and Cancer Therapeutics, The Institute of Cancer Research, Sutton, UK.

Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain. This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials. Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial. Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration. In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy.
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http://dx.doi.org/10.1016/j.ejca.2020.05.010DOI Listing
September 2020

Telomere Stress Potentiates STING-Dependent Anti-tumor Immunity.

Cancer Cell 2020 09 2;38(3):400-411.e6. Epub 2020 Jul 2.

Department of Pathology, UT Southwestern Medical Center, Dallas, TX 75390, USA.

Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8 T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.
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http://dx.doi.org/10.1016/j.ccell.2020.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494563PMC
September 2020

Immortalized normal human lung epithelial cell models for studying lung cancer biology.

Respir Investig 2020 Sep 22;58(5):344-354. Epub 2020 Jun 22.

Hamon Center for Therapeutic Oncology Research and the Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390, USA. Electronic address:

Primary cultures of human lung epithelial cells are ideal representatives of normal lung epithelial cells, and while there are certain novel approaches for the long-term culture of lung epithelial cells, the cells eventually undergo irreversible growth arrest, limiting their experimental utility, particularly the ability to widely distribute these cultures and their clonal derivatives to the broader research community. Therefore, the establishment of immortalized normal human lung epithelial cell strains has garnered considerable attention. The number and type of oncogenic changes necessary for the tumorigenic transformation of normal cells could be determined using "normal" cell lines immortalized with the simian virus 40 (SV40) large T antigen (LT). A primary report suggested that LT, human telomerase reverse transcriptase (hTERT), and oncogenic RAS transformed normal lung epithelial cells into tumorigenic cells. Since LT inactivates the tumor suppressors p53 and RB, at least four alterations would be necessary. However, the SV40 small T antigen (ST), a different oncoprotein, was also introduced simultaneously with LT in the above-mentioned study. Furthermore, the possible uncharacterized functions of LT remained largely obscure. Therefore, no definitive conclusion could be arrived in these studies. Subsequent studies used methods that did not involve the use of oncoproteins and revealed that at least five genetic changes were necessary for full tumorigenic transformation. hTERT-immortalized normal human lung epithelial cell lines established without using viral oncoproteins were also used for investigating several aspects of lung cancer, such as epithelial to mesenchymal transition and the cancer stem cell theory. The use of immortalized normal lung epithelial cell models has improved our understanding of lung cancer pathogenesis and these models can serve as valuable research tools.
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http://dx.doi.org/10.1016/j.resinv.2020.04.005DOI Listing
September 2020

Dysfunctional telomeres trigger cellular senescence mediated by cyclic GMP-AMP synthase.

J Biol Chem 2020 08 15;295(32):11144-11160. Epub 2020 Jun 15.

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double strand breaks specifically in the telomeres, ChIP, telomere immunofluorescence, fluorescence hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.
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http://dx.doi.org/10.1074/jbc.RA120.012962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415994PMC
August 2020

Chemical intervention of influenza virus mRNA nuclear export.

PLoS Pathog 2020 04 2;16(4):e1008407. Epub 2020 Apr 2.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.
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http://dx.doi.org/10.1371/journal.ppat.1008407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117665PMC
April 2020

TRA-1-60-positive/CD45 cells found in the peripheral blood of prostate cancer patients with metastatic disease - A proof-of-concept study.

Heliyon 2020 Jan 24;6(1):e03263. Epub 2020 Jan 24.

Genitourinary Oncology, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Purpose: Over 90% of all cancer related deaths are due to metastasis. However, current diagnostic tools can't reliably discriminate between invasive and localized cancers.

Patients And Methods: In this proof-of-concept study, we employed the embryonic stem cell marker TRA-1-60 (TRA+) to identify TRA + cells within the blood of prostate cancer patients and searched for TRA + cells in men with metastatic and localized cancers. We isolated whole peripheral blood mononuclear cells from 26 metastatic prostate cancer patients, from 13 patients with localized prostate cancer and from 17 healthy controls. Cells were stained for DAPI, CD45 and TRA + by immunofluorescence and imaged by epi-fluorescence microscopy. Imaged-based software was used both to identify TRA + cells, and to analyze CD45 levels in TRA+ and negative cells.

Results: We found high numbers of TRA + cells within the blood of metastatic cancer patients, whereas healthy individuals or men with localized prostate cancer showed none or very low numbers of TRA + cells. Further analysis of the CD45 levels of TRA + cells revealed a small population of TRA + cells with almost undetectable CD45 levels that were found frequently in metastatic prostate cancer patients. By excluding CD45 positive cells from the TRA + cell pool, we were able to refine the assay to be highly specific in identifying men with metastatic disease. In fact, the difference of CD45 levels between TRA+ and negative cells was a robust measure to distinguish between men with localized and metastatic prostate cancers in this small patient cohort.

Conclusions: The data suggest that metastatic prostate cancer patient have significant numbers of TRA+/CD45 cells which might represent a potential tool for diagnostic assessment in the future.
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http://dx.doi.org/10.1016/j.heliyon.2020.e03263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994489PMC
January 2020

Lung cancer progression using fast switching multiple ion beam radiation and countermeasure prevention.

Life Sci Space Res (Amst) 2020 Feb 1;24:108-115. Epub 2019 Aug 1.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Most of the research in understanding space radiation-induced cancer progression and risk assessment has been performed using mono-energetic single-ion beams. However, the space radiation environment consists of a wide variety of ion species with a various range of energies. Using the fast beam switching technology developed at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), ion species can be switched rapidly allowing investigators to use multiple ions with different energies to simulate more closely the radiation environment found in space. Here, we exposed a lung cancer susceptible mouse model (K-ras) to three sequential ion beams: Proton (H) (120 MeV/n) 20 cGy, Helium (He) (250 MeV/n) 5.0 cGy, and Silicon (Si) (300 MeV/n) 5.0 cGy with a dose rate of 0.5 cGy/min. Using three ion beams we performed whole body irradiation with a total dose of 30 cGy in two different orders: 3B-1 (H→He→Si) and 3B-2 (Si→He→H) and used 30 cGy H single-ion beam as a reference. In this study we show that whole-body irradiation with H→He→Si increases the incidence of premalignant lesions and systemic oxidative stress in mice 100 days post-irradiation more than (Si→He→H) and H only irradiation. Additionally, we observed an increase in adenomas with atypia and adenocarcinomas in H→He→Si irradiated mice but not in (Si→He→H) or H (30 cGy) only irradiated mice. When we used the H→He→Si irradiation sequence but skipped a day before exposing the mice to Si, we did not observe the increased incidence of cancer initiation and progression. We also found that a non-toxic anti-inflammatory, anti-oxidative radioprotector (CDDO-EA) reduced H→He→Si induced oxidative stress and cancer initiation almost back to baseline. Thus, exposure to H→He→Si elicits significant changes in lung cancer initiation that can be mitigated using CDDO-EA.
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http://dx.doi.org/10.1016/j.lssr.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991460PMC
February 2020

Is a Biomarker of Sensitivity to the Telomeric DNA Damage Mediator 6-Thio-2'-Deoxyguanosine.

Cancer Res 2020 03 16;80(5):929-936. Epub 2020 Jan 16.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of mRNA predicted sensitivity to 6-thio-dG and therefore could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056593PMC
March 2020

Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings.

FASEB J 2020 01 22;34(1):386-398. Epub 2019 Nov 22.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.
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http://dx.doi.org/10.1096/fj.201902376RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956733PMC
January 2020

MAP9 Loss Triggers Chromosomal Instability, Initiates Colorectal Tumorigenesis, and Is Associated with Poor Survival of Patients with Colorectal Cancer.

Clin Cancer Res 2020 02 29;26(3):746-757. Epub 2019 Oct 29.

Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK-Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong.

Purpose: Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in -knockout mice and in cell lines.

Experimental Design: We generated colon epithelial-specific -knockout mice and evaluated colorectal cancer development. Effect of knockout on colorectal cancer progression was determined in chemical or -induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC.

Results: Spontaneous colonic tumors (9.1%) were developed in colon epithelium-specific -knockout mice at 17 months, but none was observed in wild-type littermates. deletion accelerated colorectal cancer formation both in mice and azoxymethane-treated mice, and reduced survival in mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from knockout mice. In patients with colorectal cancer, was frequently silenced and its downregulation was associated with poor survival.

Conclusions: MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1611DOI Listing
February 2020

Telomere clustering drives ALT.

Aging (Albany NY) 2019 10 13;11(19):8046-8047. Epub 2019 Oct 13.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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http://dx.doi.org/10.18632/aging.102369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814596PMC
October 2019

Decellularized mice colons as models to study the contribution of the extracellular matrix to cell behavior and colon cancer progression.

Acta Biomater 2019 12 25;100:213-222. Epub 2019 Sep 25.

Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, United States. Electronic address:

Current 3D culture models to study colorectal cancer lack architectural support and signaling proteins provided by the tissue extracellular matrix (ECM) which may influence cell behavior and cancer progression. Therefore, the ability to study cancer cells in the context of a matrix that is physiologically more relevant and to understand how the ECM affects cancer progression has been understudied. To address this, we developed an ex-vivo 3D system, provided by intact wild type (WT) and colon cancer susceptible decellularized mouse colons (DMC), to support the growth of human cancer cells. DMC are free of viable cells but still contain extracellular matrix proteins including subsets of collagens. Stiffness, an important mechanical property, is also maintained in DMCs. Importantly, we observed that the DMC is permissive for cell proliferation and differentiation of a human colon cancer cell line (HT-29). Notably, the ability of cells in the WT DMC to differentiate was also greater when compared to Matrigel™, an extracellular matrix extract from a mouse tumor cell line. Additionally, we observed in invasion assays that DMC obtained from polyps from a colon cancer susceptible mouse model facilitated increased cell migration/invasion of colorectal cancer cells and immortalized non-tumor colonic epithelial cells compared to DMC from WT mice. Finally, using mass spectrometry, we identified extracellular matrix proteins that are more abundant in DMC from a colorectal cancer mouse model compared to age and sex-matched WT mice. We propose that these abundantly expressed proteins in the tumor microenvironment are potentially involved in colorectal cancer progression. STATEMENT OF SIGNIFICANCE: Decellularized matrices, when properly produced, are attractive biomaterials for tissue regeneration and replacement. We show here that the mouse decellularized matrices can also be repurposed to elucidate how the extracellular matrix influences human cell behavior and cancer progression. To do this we produce decellularized matrices, from mice colonic tissue, that have preserved tissue mechanical and structural properties. We demonstrate that the matrix better supports the differentiation of HT-29 cells, a colonic cancer cell line, compared to Matrigel™. Additionally, we show that the extracellular matrix contributes to colon cancer progression via invasion assays using extracellular matrix extracts. Finally, we use mass spectrometry to identify ECM proteins that are more abundant in colonic polyps compared to adjacent tissue regions. This model system may have therapeutic implications for colorectal cancer patients.
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http://dx.doi.org/10.1016/j.actbio.2019.09.033DOI Listing
December 2019

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution.

Genome Res 2019 11 23;29(11):1878-1888. Epub 2019 Sep 23.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas 75390, USA.

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.
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http://dx.doi.org/10.1101/gr.250480.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836731PMC
November 2019

MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer.

Genes Dev 2019 09 15;33(17-18):1236-1251. Epub 2019 Aug 15.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.
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http://dx.doi.org/10.1101/gad.327056.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719621PMC
September 2019

Infection with genotoxin-producing Salmonella enterica synergises with loss of the tumour suppressor APC in promoting genomic instability via the PI3K pathway in colonic epithelial cells.

Cell Microbiol 2019 12 26;21(12):e13099. Epub 2019 Aug 26.

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.
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http://dx.doi.org/10.1111/cmi.13099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899655PMC
December 2019

Catalysis-dependent inactivation of human telomerase and its reactivation by intracellular telomerase-activating factors (iTAFs).

J Biol Chem 2019 07 11;294(30):11579-11596. Epub 2019 Jun 11.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Human telomerase maintains genome stability by adding telomeric repeats to the ends of linear chromosomes. Although previous studies have revealed profound insights into telomerase functions, the low cellular abundance of functional telomerase and the difficulties in quantifying its activity leave its thermodynamic and kinetic properties only partially characterized. Employing a stable cell line overexpressing both the human telomerase RNA component and the N-terminally biotinylated human telomerase reverse transcriptase and using a newly developed method to count individual extension products, we demonstrate here that human telomerase holoenzymes contain fast- and slow-acting catalytic sites. Surprisingly, both active sites became inactive after two consecutive rounds of catalysis, named single-run catalysis. The fast active sites turned off ∼40-fold quicker than the slow ones and exhibited higher affinities to DNA substrates. In a dimeric enzyme, the two active sites work in tandem, with the faster site functioning before the slower one, and in the monomeric enzyme, the active sites also perform single-run catalysis. Interestingly, inactive enzymes could be reactivated by intracellular telomerase-activating factors (iTAFs) from multiple cell types. We conclude that the single-run catalysis and the iTAF-triggered reactivation serve as an unprecedented control circuit for dynamic regulation of telomerase. They endow native telomerase holoenzymes with the ability to match their total number of active sites to the number of telomeres they extend. We propose that the exquisite kinetic control of telomerase activity may play important roles in both cell division and cell aging.
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http://dx.doi.org/10.1074/jbc.RA118.007234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663873PMC
July 2019

Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52.

Genes Dev 2019 07 6;33(13-14):814-827. Epub 2019 Jun 6.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390, USA.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to type II ALT survivors.
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http://dx.doi.org/10.1101/gad.324905.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601508PMC
July 2019

Transient introduction of human telomerase mRNA improves hallmarks of progeria cells.

Aging Cell 2019 08 31;18(4):e12979. Epub 2019 May 31.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas.

Hutchinson-Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra-individual heterogeneity of expression of telomere-associated proteins DNA PKcs/Ku70/Ku80, with low-expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere-associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by β-galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.
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http://dx.doi.org/10.1111/acel.12979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612639PMC
August 2019

Design and Synthesis of TASIN Analogues Specifically Targeting Colorectal Cancer Cell Lines with Mutant Adenomatous Polyposis Coli (APC).

J Med Chem 2019 05 9;62(10):5217-5241. Epub 2019 May 9.

Despite advances in targeted anticancer therapies, there are still no small-molecule-based therapies available that specifically target colorectal cancer (CRC) development and progression, the second leading cause of cancer deaths. We previously disclosed the discovery of truncating adenomatous polyposis coli (APC)-selective inhibitor 1 (TASIN-1), a small molecule that specifically targets colorectal cancer cells lines with truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene through inhibition of cholesterol biosynthesis. Here, we report a medicinal chemistry evaluation of a collection of TASIN analogues and activity against colon cancer cell lines and an isogenic cell line pair reporting on the status of APC-dependent selectivity. A number of potent and selective analogues were identified, including compounds with good metabolic stability and pharmacokinetic properties. The compounds reported herein represent a first-in-class genotype-selective series that specifically target apc mutations present in the majority of CRC patients and serve as a translational platform toward a targeted therapy for colon cancer.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252524PMC
May 2019

In perspective: An update on telomere targeting in cancer.

Mol Carcinog 2019 09 6;58(9):1581-1588. Epub 2019 May 6.

University of Texas Southwestern, Department of Cell Biology, Dallas, Texas.

Engaging a telomere maintenance mechanism during DNA replication is essential for almost all advanced cancers. The conversion from normal and premalignant somatic cells to advanced malignant cells often results (85%-90%) from the reactivation of the functional ribonucleoprotein holoenzyme complex, referred to as telomerase. Modulation of the human telomerase reverse transcriptase (hTERT) appears to be rate limiting to produce functional telomerase and engage a telomere maintenance mechanism. The remaining 10% to 15% of cancers overcome progressively shortened telomeres by activating an alternative lengthening of telomeres (ALT) maintenance mechanism, through a DNA recombination pathway. Exploration into the specific mechanisms of telomere maintenance in cancer have led to the development of drugs such as Imetelstat (GRN163L), BIBR1532, 6-thio-dG, VE-822, and NVP-BEZ235 being investigated as therapeutic approaches for treating telomerase and ALT tumors. The successful use of 6-thio-dG (a nucleoside preferentially recognized by telomerase) that targets and uncaps telomeres in telomerase positive but not normal telomerase silent cells has recently shown impressive effects on multiple types of cancer. For example, 6-thio-dG overcomes therapy-resistant cancers in a fast-acting mechanism potentially providing an alternative or additional route of treatment for patients with cancer. In this perspective, we provide a synopsis of the current landscape of telomeres and telomerase processing in cancer development and how this new knowledge may improve outcomes for patients with cancer.
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http://dx.doi.org/10.1002/mc.23035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692182PMC
September 2019

Telomeres and telomerase: three decades of progress.

Nat Rev Genet 2019 05;20(5):299-309

Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.

Many recent advances have emerged in the telomere and telomerase fields. This Timeline article highlights the key advances that have expanded our views on the mechanistic underpinnings of telomeres and telomerase and their roles in ageing and disease. Three decades ago, the classic view was that telomeres protected the natural ends of linear chromosomes and that telomerase was a specific telomere-terminal transferase necessary for the replication of chromosome ends in single-celled organisms. While this concept is still correct, many diverse fields associated with telomeres and telomerase have substantially matured. These areas include the discovery of most of the key molecular components of telomerase, implications for limits to cellular replication, identification and characterization of human genetic disorders that result in premature telomere shortening, the concept that inhibiting telomerase might be a successful therapeutic strategy and roles for telomeres in regulating gene expression. We discuss progress in these areas and conclude with challenges and unanswered questions in the field.
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http://dx.doi.org/10.1038/s41576-019-0099-1DOI Listing
May 2019

NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells.

Oncogene 2019 04 19;38(16):2937-2952. Epub 2018 Dec 19.

School of Kinesiology, University of Michigan, 401 Washtenaw Ave., Ann Arbor Michigan, MI, 48109, USA.

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.
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http://dx.doi.org/10.1038/s41388-018-0639-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474811PMC
April 2019

Inducing rapid telomere irreparable damage in telomerase-expressing cancers.

Oncotarget 2018 Nov 9;9(88):35803-35804. Epub 2018 Nov 9.

UT Southwestern Medical Center, Department of Cell Biology, Dallas, TX, USA.

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http://dx.doi.org/10.18632/oncotarget.26317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6254679PMC
November 2018

Telomere length and telomerase activity in T cells are biomarkers of high-performing centenarians.

Aging Cell 2019 02 28;18(1):e12859. Epub 2018 Nov 28.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas.

It is generally recognized that the function of the immune system declines with increased age and one of the major immune changes is impaired T-cell responses upon antigen presentation/stimulation. Some "high-performing" centenarians (100+ years old) are remarkably successful in escaping, or largely postponing, major age-related diseases. However, the majority of centenarians ("low-performing") have experienced these pathologies and are forced to reside in long-term nursing facilities. Previous studies have pooled all centenarians examining heterogeneous populations of resting/unstimulated peripheral blood mononuclear cells (PBMCs). T cells represent around 60% of PBMC and are in a quiescence state when unstimulated. However, upon stimulation, T cells rapidly divide and exhibit dramatic changes in gene expression. We have compared stimulated T-cell responses and identified a set of transcripts expressed in vitro that are dramatically different in high- vs. low-performing centenarians. We have also identified several other measurements that are different between high- and low-performing centenarians: (a) The amount of proliferation following in vitro stimulation is dramatically greater in high-performing centenarians compared to 67- to 83-year-old controls and low-performing centenarians; (b) telomere length is greater in the high-performing centenarians; and (c) telomerase activity following stimulation is greater in the high-performing centenarians. In addition, we have validated a number of genes whose expression is directly related to telomere length and these are potential fundamental biomarkers of aging that may influence the risk and progression of multiple aging conditions.
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http://dx.doi.org/10.1111/acel.12859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351827PMC
February 2019

Proton radiation-induced cancer progression.

Life Sci Space Res (Amst) 2018 Nov 18;19:31-42. Epub 2018 Aug 18.

Department of Cell Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390, USA. Electronic address:

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-ras). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-ras mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.
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http://dx.doi.org/10.1016/j.lssr.2018.08.002DOI Listing
November 2018