IJMS, Vol. 19, Pages 608: Presence of TERT Promoter Mutations is a Secondary Event and Associates with Elongated Telomere Length in Myxoid Liposarcomas

IJMS, Vol. 19, Pages 608: Presence of TERT Promoter Mutations is a Secondary Event and Associates with Elongated Telomere Length in Myxoid Liposarcomas

International Journal of Molecular Sciences doi: 10.3390/ijms19020608

Authors: Monica S. Ventura Ferreira Martina Crysandt Till Braunschweig Edgar Jost Barbara Voss Anne-Sophie Bouillon Ruth Knuechel Tim H. Brümmendorf Fabian Beier

The occurrence of TERT promoter mutations has been well described in soft tissue sarcomas (STS). However, the biological role of these mutations as well as their impact on telomere length in STS is still unclear. We analyzed 116 patient samples diagnosed with 22 distinct histological subtypes of bone and STS for the occurrence of TERT promoter mutations by Sanger sequencing. We observed TERT promoter mutations at an overall frequency of 9.5% distributed over 7 different sarcoma subtypes. Except for one chondrosarcoma case harboring a C250T mutation, all other mutations were detected at location C228T. By far the far highest frequency of TERT promoter mutations was found in myxoid liposarcoma (MLS) (4 out of 9 cases studied, i.e., 44%). Assessment of telomere length from tumor biopsies revealed that TERT promoter-mutated MLSs had significantly fewer shortened telomeres in comparison to TERT wildtype MLSs. Based on the frequency of TERT promoter mutations and the elongated telomere length in mutated compared to wildtype MLS, we hypothesize that occurrence of TERT promoter mutations has a pivotal role in the disease progression as a secondary genetic event at a time when tumor cells face the need for telomere elongation to allow further proliferation.

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IJMS, Vol. 19, Pages 607: Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells

IJMS, Vol. 19, Pages 607: Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells

International Journal of Molecular Sciences doi: 10.3390/ijms19020607

Authors: Michal Sarna Andrzej Zadlo Barbara Czuba-Pelech Krystyna Urbanska

Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells’ metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells’ metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of melanoma.

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IJMS, Vol. 19, Pages 609: The Significance of MMP-1 in EGFR-TKI–Resistant Lung Adenocarcinoma: Potential for Therapeutic Targeting

IJMS, Vol. 19, Pages 609: The Significance of MMP-1 in EGFR-TKI–Resistant Lung Adenocarcinoma: Potential for Therapeutic Targeting

International Journal of Molecular Sciences doi: 10.3390/ijms19020609

Authors: Ryoko Saito Yasuhiro Miki Naoya Ishida Chihiro Inoue Masayuki Kobayashi Shuko Hata Hisafumi Yamada-Okabe Yoshinori Okada Hironobu Sasano

Epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance is one of the most important problems in lung cancer therapy. Lung adenocarcinoma with EGFR-TKI resistance was reported to have higher abilities of invasion and migration than cancers sensitive to EGFR-TKI, but the function of matrix metalloproteinases (MMPs) has not been explored in EGFR-TKI–resistant lung adenocarcinoma. This study aims to clarify the significance of MMP-1 in EGFR-TKI–resistant lung adenocarcinoma. From the results of in vitro studies of migration and invasion assays using EGFR-TKI–sensitive and –resistant cell lines and phosphorylation antibody arrays using EGF and rapamycin, we first demonstrate that overexpression of MMP-1, which might follow activation of a mammalian target of rapamycin (mTOR) pathway, plays an important role in the migration and invasion abilities of EGFR-TKI–resistant lung adenocarcinoma. Additionally, immunohistochemical studies using 89 cases of lung adenocarcinoma demonstrate that high expression of MMP-1 is significantly correlated with poor prognosis and factors such as smoking history and the subtype of invasive mucinous adenocarcinoma. These are consistent with the results of this in vitro study. To conclude, this study provides insights into the development of a possible alternative therapy manipulating MMP-1 and the mTOR signaling pathway in EGFR-TKI–resistant lung adenocarcinoma.

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IJMS, Vol. 19, Pages 606: Telomere Length Maintenance in Cancer: At the Crossroad between Telomerase and Alternative Lengthening of Telomeres (ALT)

IJMS, Vol. 19, Pages 606: Telomere Length Maintenance in Cancer: At the Crossroad between Telomerase and Alternative Lengthening of Telomeres (ALT)

International Journal of Molecular Sciences doi: 10.3390/ijms19020606

Authors: Marco De Vitis Francesco Berardinelli Antonella Sgura

Eukaryotic cells undergo continuous telomere shortening as a consequence of multiple rounds of replications. During tumorigenesis, cells have to acquire telomere DNA maintenance mechanisms (TMMs) in order to counteract telomere shortening, to preserve telomeres from DNA damage repair systems and to avoid telomere-mediated senescence and/or apoptosis. For this reason, telomere maintenance is an essential step in cancer progression. Most human tumors maintain their telomeres expressing telomerase, whereas a lower but significant proportion activates the alternative lengthening of telomeres (ALT) pathway. However, evidence about the coexistence of ALT and telomerase has been found both in vivo in the same cancer populations and in vitro in engineered cellular models, making the distinction between telomerase- and ALT-positive tumors elusive. Indeed, after the development of drugs able to target telomerase, the capability for some cancer cells to escape death, switching from telomerase to ALT, was highlighted. Unfortunately, to date, the mechanism underlying the possible switching or the coexistence of telomerase and ALT within the same cell or populations is not completely understood and different factors could be involved. In recent years, different studies have tried to shed light on the complex regulation network that controls the transition between the two TMMs, suggesting a role for embryonic cancer origin, epigenetic modifications, and specific genes activation—both in vivo and in vitro. In this review, we examine recent findings about the cancer-associated differential activation of the two known TMMs and the possible factors implicated in this process. Furthermore, some studies on cancers are also described that did not display any TMM.

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IJMS, Vol. 19, Pages 605: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

IJMS, Vol. 19, Pages 605: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

International Journal of Molecular Sciences doi: 10.3390/ijms19020605

Authors: Wei Zhang Terunao Takahara Takuya Achiha Hideki Shibata Masatoshi Maki

NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization.

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Evaluating the impact of diabetes and diabetic cardiomyopathy rat heart on the outcome of ischemia-reperfusion associated oxidative stress

Publication date: Available online 17 February 2018
Source:Free Radical Biology and Medicine
Author(s): A. Mahalakshmi, Gino A. Kurian
Earlier literature underlines that oxidative stress plays a major role in the pathology of myocardial ischemia-reperfusion (I/R) injury, diabetic cardiomyopathy (DCM), diabetes mellitus (DM), fibrosis and hypertrophy which could adversely affect the normal cardiac function. However, the contributory role of oxidative stress in I/R pathology of heart with pre-existing abnormalities or diseases like DM and DCM remains to be explored. I/R injury was induced in normal (normal diet), DM (normal diet + streptozotocin: multiple low dose of 30mg/kg) and DCM (high fat diet (40% fat) + streptozotocin: multiple low dose of 30mg/kg) rat hearts using Langendorff isolated heart perfusion apparatus. Cardiac physiological recovery after I/R was assessed by hemodynamic parameters like LVDP, and LVSP, whereas cardiac injury was measured by tissue infarct size, and apoptosis, LDH, and CK release in coronary effluent. The oxidative stress was evaluated in myocardial homogenate, mitochondrial subpopulation, and microsomes. Reperfusing the ischemic DCM heart significantly deteriorated cardiac physiological recovery and elevated the cardiac injury (infarct size: 60%), compared to the control. But in DM heart, physiological recovery was prominent in the initial phase of reperfusion but deteriorated towards the end of reperfusion, supported by less infarct size. In addition, elevated lipid peroxidation (70% in DCM-I/R vs Sham) and impaired antioxidant enzymes (% decline vs Sham: GSH- 56% (DM), 63% (DCM); Catalase- 58% (DM), 35% (DCM); GPx- 19% (DM), 27% (DCM) and GR- 28% (DCM)) was observed in myocardial tissue from both DM and DCM. Interestingly, upon reperfusion, only normal heart showed significant deterioration in the antioxidant defense system. Collectively these results demonstrated that I/R induced oxidative stress is minimal in DM and DCM rat heart, despite high infarct size and low cardiac performance. This may be due to the prior adaptive modification in the antioxidant system associated with disease pathology.

Graphical abstract

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PINK1 Protects Auditory Hair Cells and Spiral Ganglion Neurons from Cisplatin-induced Ototoxicity via Inducing Autophagy and Inhibiting JNK Signaling Pathway

Publication date: Available online 17 February 2018
Source:Free Radical Biology and Medicine
Author(s): Qianqian Yang, Gaoying Sun, Haiyan Yin, Hongrui Li, Zhixin Cao, Jinghan Wang, Meijuan Zhou, Haibo Wang, Jianfeng Li
Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) gene encodes a serine/threonine kinase, which acts as a molecular sensor of mitochondrial health necessary for mitochondrial quality control. The present study was designed to examine whether PINK1 expressed in C57BL/6 murine cochlea and HEI-OC1 cells and, if so, to investigate the possible mechanisms underlying the action of PINK1 in cisplatin-induced death of sensory hair cells (HCs) and spiral ganglion neurons (SGNs) in vitro. The expression pattern of PINK1, formation of parkin particles, and autophagy were determined by immunofluorescent staining. The expressions of PINK1, LC3B, cleaved-caspase 3 and p-JNK were measured by Western blotting. The levels of reactive oxygen species (ROS) were evaluated by DCFH-DA and Mito-Sox Red staining. The mitochondrial membrane potential was detected by Tetramethylrhodamine methyl ester perchlorate (TMRM) and Rhodamine 123. Cell viability and apoptosis were examined by CCK8 assay, TUNEL staining and Annexin V Apoptosis Detection Kit, respectively. We found that PINK1 was widely expressed in the cytoplasm in HCs, SGNs, stria vascularis of C57BL/6 cochlea and HEI-OC1 cells and, notably, the expression level in cochlear HCs and SGNs of postnatal day 4 (P4) mice was higher than that in adult mice. Moreover, treatment with 30μM cisplatin elicited the formation of ROS, which, in turn, led to PINK1 activation, parkin recruitment, autophagy formation and JNK pathway relevant to apoptosis in HEI-OC1 cells, HCs, and SGNs. Meanwhile, co-treatment with ROS scavenger N-acetyl-L-cysteine (NAC) or H2O2 consumer catalase-polyethylene glycol (PEG-catalase) inhibited parkin recruitment, alleviated autophagy formation, and mitigated JNK pathway related apoptosis. In addition, PINK1 silencing resulted in a lower level of autophagy, but, a higher mortality in HEI-OC1 cells treated with cisplatin. Taken together, data from this work reveal that PINK1 possesses the protective effect via induction of autophagy and resistance of apoptosis under cisplatin stimulus in sensory HCs and SGNs, implying that PINK1 might serve as an important regulator of cisplatin-elicited ototoxicity.

Graphical abstract

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