bims-ectoca 2024-07-14 papers

bims-ectoca

Biomed News

on Epigenetic control of tolerance in cancer

Issue of 2024‒07‒14
thirteen papers selected by
Ankita Daiya, OneCell Diagnostics Inc.

Transcriptomic Analysis Reveals Early Alterations Associated with Intrinsic Resistance to Targeted Therapy in Lung Adenocarcinoma Cell Lines.
Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.
YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity.
To Erase or Not to Erase: Non-Canonical Catalytic Functions and Non-Catalytic Functions of Members of Histone Lysine Demethylase Families.
Challenges of Regulated Cell Death: Implications for Therapy Resistance in Cancer.
Characterization of spinal hemangioblastomas in patients with and without von Hippel-Lindau, and YAP expression.
Inhibition of Histone Deacetylase Activity Increases Cisplatin Efficacy to Eliminate Metastatic Cells in Pediatric Liver Cancers.
GLP and G9a histone methyltransferases as potential therapeutic targets for lymphoid neoplasms.
LAMTOR5-AS1 Regulates Chemotherapy-induced Oxidative Stress via the miR-34a-3p/SIRT1/HNF4A Axis in Osteosarcoma Cells.
Role of Wnt/β-catenin pathway in cancer drug resistance: Insights into molecular aspects of major solid tumors.
Role of diffusion and reaction of the constituents in spreading of histone modification marks.
LIMK1 promotes the development of cervical cancer by up-regulating the ROS/Src-FAK/cofilin signaling pathway.
Functional consequences of a p53-MDM2-p21 incoherent feedforward loop.

Cancers (Basel). 2024 Jul 08. pii: 2490. [Epub ahead of print]16(13):

Transcriptomic Analysis Reveals Early Alterations Associated with Intrinsic Resistance to Targeted Therapy in Lung Adenocarcinoma Cell Lines.

Mario Perez-Medina, Jose S Lopez-Gonzalez, Jesus J Benito-Lopez, Santiago Ávila-Ríos, Maribel Soto-Nava, Margarita Matias-Florentino, Alfonso Méndez-Tenorio, Miriam Galicia-Velasco, Rodolfo Chavez-Dominguez, Sergio E Meza-Toledo, Dolores Aguilar-Cazares.

  Lung adenocarcinoma is the most prevalent form of lung cancer, and drug resistance poses a significant obstacle in its treatment. This study aimed to investigate the overexpression of long non-coding RNAs (lncRNAs) as a mechanism that promotes intrinsic resistance in tumor cells from the onset of treatment. Drug-tolerant persister (DTP) cells are a subset of cancer cells that survive and proliferate after exposure to therapeutic drugs, making them an essential object of study in cancer treatment. The molecular mechanisms underlying DTP cell survival are not fully understood; however, long non-coding RNAs (lncRNAs) have been proposed to play a crucial role. DTP cells from lung adenocarcinoma cell lines were obtained after single exposure to tyrosine kinase inhibitors (TKIs; erlotinib or osimertinib). After establishing DTP cells, RNA sequencing was performed to investigate the differential expression of the lncRNAs. Some lncRNAs and one mRNA were overexpressed in DTP cells. The clinical relevance of lncRNAs was evaluated in a cohort of patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA). RT-qPCR validated the overexpression of lncRNAs and mRNA in the residual DTP cells and LUAD biopsies. Knockdown of these lncRNAs increases the sensitivity of DTP cells to therapeutic drugs. This study provides an opportunity to investigate the involvement of lncRNAs in the genetic and epigenetic mechanisms that underlie intrinsic resistance. The identified lncRNAs and CD74 mRNA may serve as potential prognostic markers or therapeutic targets to improve the overall survival (OS) of patients with lung cancer.

Keywords:  CD74; drug-tolerant persister (DTP) cells; erlotinib; intrinsic resistance; lung adenocarcinoma; osimertinib; senescent cells; tyrosine kinase inhibitors (TKIs)

DOI:  https://doi.org/10.3390/cancers16132490
bioRxiv. 2024 Jun 25. pii: 2024.06.24.600310. [Epub ahead of print]

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.

Monika Dhankhar, Zixian Guo, Aayush Kant, Ramin Basir, Rohit Joshi, Su Chin Heo, Robert L Mauck, Melike Lakadamyali, Vivek B Shenoy.

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2 β ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

DOI: https://doi.org/10.1101/2024.06.24.600310
bioRxiv. 2024 Jun 27. pii: 2024.06.21.600079. [Epub ahead of print]

YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity.

Xu Li, Yong Suc Cho, Yuchen Liu, Yingzi Yang, Shu Zhuo, Jin Jiang.

Hippo pathway functions as a tumor suppressor pathway by inhibiting the oncogenic potential of pathway effectors YAP/TAZ. However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). Here we show that YAP blocks NF-κB signaling in ccRCC to inhibit cancer cell growth. Mechanistically, YAP inhibits the expression of ZHX2, a critical p65 co-factor in ccRCC. Furthermore, YAP competes with ZHX2 for binding to p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and p65, leading to diminished NF-κB target gene expression. Pharmacological inhibition of Hippo/MST1/2 blocked NF-κB transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by ZHX2/p65 overexpression. Our study uncovers a novel crosstalk between the Hippo and NF-κB pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

DOI: https://doi.org/10.1101/2024.06.21.600079
Int J Mol Sci. 2024 Jun 24. pii: 6900. [Epub ahead of print]25(13):

To Erase or Not to Erase: Non-Canonical Catalytic Functions and Non-Catalytic Functions of Members of Histone Lysine Demethylase Families.

Elena Di Nisio, Valeria Manzini, Valerio Licursi, Rodolfo Negri.

  Histone lysine demethylases (KDMs) play an essential role in biological processes such as transcription regulation, RNA maturation, transposable element control, and genome damage sensing and repair. In most cases, their action requires catalytic activities, but non-catalytic functions have also been shown in some KDMs. Indeed, some strictly KDM-related proteins and some KDM isoforms do not act as histone demethylase but show other enzymatic activities or relevant non-enzymatic functions in different cell types. Moreover, many studies have reported on functions potentially supported by catalytically dead mutant KDMs. This is probably due to the versatility of the catalytical core, which can adapt to assume different molecular functions, and to the complex multi-domain structure of these proteins which encompasses functional modules for targeting histone modifications, promoting protein-protein interactions, or recognizing nucleic acid structural motifs. This rich modularity and the availability of multiple isoforms in the various classes produced variants with enzymatic functions aside from histone demethylation or variants with non-catalytical functions during the evolution. In this review we will catalog the proteins with null or questionable demethylase activity and predicted or validated inactive isoforms, summarizing what is known about their alternative functions. We will then go through some experimental evidence for the non-catalytical functions of active KDMs.

Keywords:  cancer epigenetics; catalytic-independent functions; histone demethylases; histone modifications; protein isoforms

DOI:  https://doi.org/10.3390/ijms25136900
Cells. 2024 Jun 22. pii: 1083. [Epub ahead of print]13(13):

Challenges of Regulated Cell Death: Implications for Therapy Resistance in Cancer.

Maria D'Amico, Francesca De Amicis.

  Regulated cell death, a regulatory form of cell demise, has been extensively studied in multicellular organisms. It plays a pivotal role in maintaining organismal homeostasis under normal and pathological conditions. Although alterations in various regulated cell death modes are hallmark features of tumorigenesis, they can have divergent effects on cancer cells. Consequently, there is a growing interest in targeting these mechanisms using small-molecule compounds for therapeutic purposes, with substantial progress observed across various human cancers. This review focuses on summarizing key signaling pathways associated with apoptotic and autophagy-dependent cell death. Additionally, it explores crucial pathways related to other regulated cell death modes in the context of cancer. The discussion delves into the current understanding of these processes and their implications in cancer treatment, aiming to illuminate novel strategies to combat therapy resistance and enhance overall cancer therapy.

Keywords:  apoptosis; autophagy; drug resistance; ferroptosis; immunogenic; pyroptosis

DOI:  https://doi.org/10.3390/cells13131083
Rev Esp Patol. 2024 Jul-Sep;57(3):pii: S1699-8855(24)00034-5. [Epub ahead of print]57(3): 160-168

Characterization of spinal hemangioblastomas in patients with and without von Hippel-Lindau, and YAP expression.

Ana-Laura Calderón-Garcidueñas, Steven-Andrés Piña-Ballantyne, Eunice-Jazmín Espinosa-Aguilar, Rebeca de Jesús Ramos-Sánchez.

  INTRODUCTION: Hemangioblastoma (HB) is a benign tumor of the central nervous system, associated with von Hippel-Lindau disease (VHL), or sporadic. The aim of this study was to compare and examine the clinical-pathological profile of patients with spinal hemangioblastoma and YAP expression.METHODS: A retrospective, descriptive, comparative study. All patients who underwent surgery for spinal HB between 2016 and 2023 were included. Clinical and radiological data were collected and analyzed. An immunohistochemistry panel including NeuN, neurofilaments (NF), and YAP-1, was performed.
RESULTS: Nine patients were studied, six women and three men. Four patients had previously diagnosed VHL. The tumor location included: four cervical (44.44%), two thoracic (22.22%), two pontine with cervical extension (22.22%) and one patient with two lesions, one cervical and one thoracic (11.11%). Non-significant clinical differences were identified between VHL and sporadic patients. Imaging evidenced seven extramedullary and three intramedullary tumors. Histologically, intra-tumoral and perivascular axonal tracts were observed in all cases. One third of the tumors (two with VHL and one sporadic) presented extramedullary hematopoiesis. Seven cases (77.8%) expressed nuclear YAP (three with VHL and four sporadic HBs). The surgical outcome was good and only one patient with VHL undergoing subtotal resection had recurrence.
CONCLUSIONS: Spinal HBs can be associated with VHL or be sporadic. To the best of our knowledge, this is the first study to describe YAP expression in HB. It is important to investigate the involvement of the Hippo pathway in HBs as a possible therapeutic target.

Keywords:  Enfermedad de von Hippel-Lindau; Esporádico; Hemangioblastoma; Médula espinal; Spinal cord; Sporadic; Yes-associated protein; von Hippel-Lindau disease

DOI:  https://doi.org/10.1016/j.patol.2024.03.002
Cancers (Basel). 2024 Jun 22. pii: 2300. [Epub ahead of print]16(13):

Inhibition of Histone Deacetylase Activity Increases Cisplatin Efficacy to Eliminate Metastatic Cells in Pediatric Liver Cancers.

Ruhi Gulati, Yasmeen Fleifil, Katherine Jennings, Alex Bondoc, Greg Tiao, James Geller, Lubov Timchenko, Nikolai Timchenko.

  The pediatric liver cancers, hepatoblastoma and hepatocellular carcinoma, are dangerous cancers which often spread to the lungs. Although treatments with cisplatin significantly improve outcomes, cisplatin may not eliminate metastasis-initiating cells. Our group has recently shown that the metastatic microenvironments of hepatoblastoma contain Cancer Associated Fibroblasts (CAFs) and neuron-like cells, which initiate cancer spread from liver to lungs. In this study, we found that these cells express high levels of HDAC1; therefore, we examined if histone deacetylase inhibition improves cisplatin anti-proliferative effects and reduces the formation of tumor clusters in pediatric liver cancer metastatic microenvironments.METHODS: New cell lines were generated from primary hepatoblastoma liver tumors (hbl) and lung metastases (LM) of HBL patients. In addition, cell lines were generated from hepatocellular neoplasm, not otherwise specified (HCN-NOS) tumor samples, and hcc cell lines. Hbl, LM and hcc cells were treated with cisplatin, SAHA or in combination. The effect of these drugs on the number of cells, formation of tumor clusters and HDAC1-Sp5-p21 axis were examined.
RESULTS: Both HBL and HCC tissue specimens have increased HDAC1-Sp5 pathway activation, recapitulated in cell lines generated from the tumors. HDAC inhibition with vorinostat (SAHA) increases cisplatin efficacy to eliminate CAFs in hbl and in hcc cell lines. Although the neuron-like cells survive the combined treatments, proliferation was inhibited. Notably, combining SAHA with cisplatin overcame cisplatin resistance in an LM cell line from an aggressive case with multiple metastases. Underlying mechanisms of this enhanced inhibition include suppression of the HDAC1-Sp5 pathway and elevation of an inhibitor of proliferation p21. Similar findings were found with gemcitabine treatments suggesting that elimination of proliferative CAFs cells is a key event in the inhibition of mitotic microenvironment.
CONCLUSIONS: Our studies demonstrate the synergistic benefits of HDAC inhibition and cisplatin to eliminate metastasis-initiating cells in pediatric liver cancers.

Keywords:  HDAC; Sp5; cisplatin; hepatoblastoma; hepatocellular carcinoma; metastases

DOI:  https://doi.org/10.3390/cancers16132300
Cancer Cell Int. 2024 Jul 12. 24(1): 243

GLP and G9a histone methyltransferases as potential therapeutic targets for lymphoid neoplasms.

Amandda Évelin Silva-Carvalho, Luma Dayane Carvalho Filiú-Braga, Gabriela Muller Reche Bogéa, Alan Jhones Barbosa de Assis, Fábio Pittella-Silva, Felipe Saldanha-Araujo.

  Histone methyltransferases (HMTs) are enzymes that regulate histone methylation and play an important role in controlling transcription by altering the chromatin structure. Aberrant activation of HMTs has been widely reported in certain types of neoplastic cells. Among them, G9a/EHMT2 and GLP/EHMT1 are crucial for H3K9 methylation, and their dysregulation has been associated with tumor initiation and progression in different types of cancer. More recently, it has been shown that G9a and GLP appear to play a critical role in several lymphoid hematologic malignancies. Importantly, the key roles played by both enzymes in various diseases made them attractive targets for drug development. In fact, in recent years, several groups have tried to develop small molecule inhibitors targeting their epigenetic activities as potential anticancer therapeutic tools. In this review, we discuss the physiological role of GLP and G9a, their oncogenic functions in hematologic malignancies of the lymphoid lineage, and the therapeutic potential of epigenetic drugs targeting G9a/GLP for cancer treatment.

Keywords:  Acute lymphoblastic leukemia; Chronic lymphoblastic leukemia; G9a; GLP; Lymphoid neoplasms; Multiple myeloma

DOI:  https://doi.org/10.1186/s12935-024-03441-y
Curr Med Chem. 2024 Jul 11.

LAMTOR5-AS1 Regulates Chemotherapy-induced Oxidative Stress via the miR-34a-3p/SIRT1/HNF4A Axis in Osteosarcoma Cells.

Fangfang Zhao, Chao Wei, Youguang Pu, Chunbao Zang.

  INTRODUCTION: Osteosarcoma (OS) drug resistance often leads to a poor prognosis. Recent evidence suggests that long non-coding RNAs play a crucial role in regulating tumor drug resistance.METHOD: This study aims to investigate the involvement of lncRNA LAMTOR5-AS1 in OS. RNA-seq and qRT-PCR were performed, and the relationship between LAMTOR5- AS1, miR-34a-3p, SIRT1, and HNF4A was determined using Dual-luciferase reporter assays and RNA immunoprecipitation assays. Gain- and loss-of-function assays were measured using CCK-8, cell proliferation, and colony formation assays.
RESULT: The study found that the dysregulated LAMTOR5-AS1 acts as a competing endogenous RNA (ceRNA) and competitively protects the HNF4A mRNA 3' UTR from miR-34a-3p. In addition, in vitro functional studies showed that HNF4A can physically interact with SIRT1 to synergistically inhibit osteosarcoma drug resistance. The study found that LAMTOR5-AS1 regulates drug resistance in osteosarcoma through the miR-34a-3p/HNF4A or miR-34a-3p/SIRT1/HNF4A axis.
CONCLUSION: These findings offer new insights into lncRNA-mediated drug resistance in cancer and may serve as potential biomarkers for cancer therapy.

Keywords:  LAMTOR5-AS1; Osteosarcoma; drug-resistance; long noncoding RNAs; miR-34a-3p.

DOI:  https://doi.org/10.2174/0109298673316534240708111058
Biochem Biophys Res Commun. 2024 Jul 04. pii: S0006-291X(24)00884-2. [Epub ahead of print]729 150348

Role of Wnt/β-catenin pathway in cancer drug resistance: Insights into molecular aspects of major solid tumors.

Charudatt Samant, Ramesh Kale, K Sreedhara Ranganath Pai, Krishnadas Nandakumar, Mandar Bhonde.

  Adaptive resistance to conventional and targeted therapies remains one of the major obstacles in the effective management of cancer. Aberrant activation of key signaling mechanisms plays a pivotal role in modulating resistance to drugs. An evolutionarily conserved Wnt/β-catenin pathway is one of the signaling cascades which regulate resistance to drugs. Elevated Wnt signaling confers resistance to anticancer therapies, either through direct activation of its target genes or via indirect mechanisms and crosstalk over other signaling pathways. Involvement of the Wnt/β-catenin pathway in cancer hallmarks like inhibition of apoptosis, promotion of invasion and metastasis and cancer stem cell maintenance makes this pathway a potential target to exploit for addressing drug resistance. Accumulating evidences suggest a critical role of Wnt/β-catenin pathway in imparting resistance across multiple cancers including PDAC, NSCLC, TNBC, etc. Here we present a comprehensive assessment of how Wnt/β-catenin pathway mediates cancer drug resistance in majority of the solid tumors. We take a deep dive into the Wnt/β-catenin signaling-mediated modulation of cellular and downstream molecular mechanisms and their impact on cancer resistance.

Keywords:  Cancer resistance; NSCLC; PDAC; Stem cells; Wnt/β-catenin

DOI:  https://doi.org/10.1016/j.bbrc.2024.150348
PLoS Comput Biol. 2024 Jul 11. 20(7): e1012235

Role of diffusion and reaction of the constituents in spreading of histone modification marks.

Vinoth Manivannan, Mandar M Inamdar, Ranjith Padinhateeri.

Cells switch genes ON or OFF by altering the state of chromatin via histone modifications at specific regulatory locations along the chromatin polymer. These gene regulation processes are carried out by a network of reactions in which the histone marks spread to neighboring regions with the help of enzymes. In the literature, this spreading has been studied as a purely kinetic, non-diffusive process considering the interactions between neighboring nucleosomes. In this work, we go beyond this framework and study the spreading of modifications using a reaction-diffusion (RD) model accounting for the diffusion of the constituents. We quantitatively segregate the modification profiles generated from kinetic and RD models. The diffusion and degradation of enzymes set a natural length scale for limiting the domain size of modification spreading, and the resulting enzyme limitation is inherent in our model. We also demonstrate the emergence of confined modification domains without the explicit requirement of a nucleation site. We explore polymer compaction effects on spreading and show that single-cell domains may differ from averaged profiles. We find that the modification profiles from our model are comparable with existing H3K9me3 data of S. pombe.

DOI: https://doi.org/10.1371/journal.pcbi.1012235
Aging (Albany NY). 2024 Jul 05. 16

LIMK1 promotes the development of cervical cancer by up-regulating the ROS/Src-FAK/cofilin signaling pathway.

Yajing Jia, Yongping Li, Naiyi Du, Wei Zhao, Yakun Liu.

  OBJECTIVE: In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression.METHODS: The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays.
RESULTS: Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells.
CONCLUSIONS: LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.

Keywords:  LIMK1; Src; cervical cancer; oxidative stress

DOI:  https://doi.org/10.18632/aging.206007
bioRxiv. 2024 Jun 29. pii: 2024.06.25.600070. [Epub ahead of print]

Functional consequences of a p53-MDM2-p21 incoherent feedforward loop.

Jamie A Dean, Jose Reyes, Michael Tsabar, Ashwini Jambhekar, Galit Lahav, Franziska Michor.

Genetically identical cells can respond heterogeneously to cancer therapy, with a subpopulation of cells often entering a temporarily arrested treatment-tolerant state before repopulating the tumor. To investigate how heterogeneity in the cell cycle arrest protein p21 arises, we imaged the dynamics of p21 transcription and protein expression along with those of p53, its transcriptional regulator, in single cells using live cell fluorescence microscopy. Surprisingly, we found that the rate of p21 transcription depends on the change in p53 rather than its absolute level. Through combined theoretical and experimental modeling, we determined that p21 transcription is governed by an incoherent feedforward loop mediated by MDM2. This network architecture facilitates rapid induction of p21 expression and variability in p21 transcription. Abrogating the feedforward loop overcomes rapid S-phase p21 degradation, with cells transitioning into a quiescent state that transcriptionally resembles a treatment-tolerant persister state. Our findings have important implications for therapeutic strategies based on activating p53.

DOI: https://doi.org/10.1101/2024.06.25.600070