Wild-Type KRAS Allele Effects on Druggable Targets in KRAS Mutant Lung Adenocarcinomas

KRAS mutations are one of the most common oncogenic drivers in non-small cell lung cancer (NSCLC) and in lung adenocarcinomas in particular. Development of therapeutics targeting KRAS has been incredibly challenging, prompting indirect inhibition of downstream targets such as MEK and ERK. Such inhibitors, unfortunately, come with limited clinical efficacy, and therefore the demand for developing novel therapeutic strategies remains an urgent need for these patients. Exploring the influence of wild-type (WT) KRAS on druggable targets can uncover new vulnerabilities for the treatment of KRAS mutant lung adenocarcinomas. Using commercially available KRAS mutant lung adenocarcinoma cell lines, we explored the influence of WT KRAS on signaling networks and druggable targets. Expression and/or activation of 183 signaling proteins, most of which are targets of FDA-approved drugs, were captured by reverse-phase protein microarray (RPPA). Selected findings were validated on a cohort of 23 surgical biospecimens using the RPPA. Kinase-driven signatures associated with the presence of the KRAS WT allele were detected along the MAPK and AKT/mTOR signaling pathway and alterations of cell cycle regulators. FoxM1 emerged as a potential vulnerability of tumors retaining the KRAS WT allele both in cell lines and in the clinical samples. Our findings suggest that loss of WT KRAS impacts on signaling events and druggable targets in KRAS mutant lung adenocarcinomas.

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Frequency and Spectrum of KRAS Mutations in Moroccan Patients with Lung Adenocarcinoma

ISRN Oncology ◽

10.1155/2014/192493 ◽

2014 ◽

Vol 2014 ◽

pp. 1-4 ◽

Cited By ~ 1

Author(s):

Ibrahim Elghissassi ◽

Hanane Inrhaoun ◽

Anwar Boukir ◽

Fouad Kettani ◽

Lamia Gamra ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Kras Mutation ◽

Mutation Frequency ◽

Asian Population ◽

Wild Type ◽

North African ◽

Kras Mutations ◽

Exon 2 ◽

Lung Adenocarcinomas ◽

Moroccan Patients

Background. In lung adenocarcinoma, the frequency of KRAS mutations is ethnicity dependent with a higher proportion in African Americans and white Caucasians than in Asians. The prevalence of these mutations among North Africans patients is unknown. The objective of this study was to report the frequency and spectrum of KRAS mutations in a group of Moroccan lung adenocarcinoma patients. Methods. Tumor specimens from 117 Moroccan patients with lung adenocarcinoma were selected to determine frequency and spectrum of KRAS mutations. KRAS mutations in codons 12 and 13 of exon 2 were analyzed using conventional DNA sequencing. Results. The overall frequency of the KRAS mutations was 9% (11/117). In the population with KRAS mutations, there was a trend towards more male (P=0.06) and more smokers (P=0.08) compared to patients with wild type KRAS. KRAS mutations were located at codon 12 in 10 out of 11 patients (91%). The G12C mutation was the most frequent KRAS mutation (73%). Conclusion. This is the first study to date examining the frequency and spectrum of KRAS mutations in lung adenocarcinomas in North African and Arab populations. KRAS mutation frequency in Moroccan patients was comparable with the frequency observed in East-Asian population. KRAS mutations are more likely observed in males and smokers and to be transversions. Further studies, in larger numbers of patients, are needed to confirm these findings.

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Contribution of nuclear BCL10 expression to tumor progression and poor prognosis of advanced and/or metastatic pancreatic ductal adenocarcinoma by activating NF-κB-related signaling

Cancer Cell International ◽

10.1186/s12935-021-02143-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Sung-Hsin Kuo ◽

Shih-Hung Yang ◽

Ming-Feng Wei ◽

Hsiao-Wei Lee ◽

Yu-Wen Tien ◽

...

Keyword(s):

Cell Cycle ◽

Tumor Progression ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Poor Prognosis ◽

Cyclin B1 ◽

Ductal Adenocarcinoma ◽

Clinical Samples ◽

Wild Type ◽

Pdac Cell

Abstract Background We previously demonstrated that nuclear BCL10 translocation participates in the instigation of NF-κB in breast cancer and lymphoma cell lines. In this study, we assessed whether nuclear BCL10 translocation is clinically significant in advanced and metastatic pancreatic ductal adenocarcinoma (PDAC). Method and materials We analyzed the expression of BCL10-, cell cycle-, and NF-κB- related signaling molecules, and the DNA-binding activity of NF-κB in three PDAC cell lines (mutant KRAS lines: PANC-1 and AsPC-1; wild-type KRAS line: BxPC-3) using BCL10 short hairpin RNA (shBCL10). To assess the anti-tumor effect of BCL10 knockdown in PDAC xenograft model, PANC-1 cells treated with or without shBCL10 transfection were inoculated into the flanks of mice. We assessed the expression patterns of BCL10 and NF-κB in tumor cells in 136 patients with recurrent, advanced, and metastatic PDAC using immunohistochemical staining. Results We revealed that shBCL10 transfection caused cytoplasmic translocation of BCL10 from the nuclei, inhibited cell viability, and enhanced the cytotoxicities of gemcitabine and oxaliplatin in three PDAC cell lines. Inhibition of BCL10 differentially blocked cell cycle progression in PDAC cell lines. Arrest at G1 phase was noted in wild-type KRAS cell lines; and arrest at G2/M phase was noted in mutant KRAS cell lines. Furthermore, shBCL10 transfection downregulated the expression of phospho-CDC2, phospho-CDC25C, Cyclin B1 (PANC-1), Cyclins A, D1, and E, CDK2, and CDK4 (BxPC-3), p-IκBα, nuclear expression of BCL10, BCL3, and NF-κB (p65), and attenuated the NF-κB pathway activation and its downstream molecule, c-Myc, while inhibition of BCL10 upregulated expression of p21, and p27 in both PANC-1 and BxPC-3 cells. In a PANC-1-xenograft mouse model, inhibition of BCL10 expression also attenuated the tumor growth of PDAC. In clinical samples, nuclear BCL10 expression was closely associated with nuclear NF-κB expression (p < 0.001), and patients with nuclear BCL10 expression had the worse median overall survival than those without nuclear BCL10 expression (6.90 months versus 9.53 months, p = 0.019). Conclusion Nuclear BCL10 translocation activates NF-κB signaling and contributes to tumor progression and poor prognosis of advanced/metastatic PDAC.

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Ultra Rapid, One Step Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Amplification Reaction.

Blood ◽

10.1182/blood.v120.21.2515.2515 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2515-2515

Author(s):

Elena D'agostini ◽

Giulia Minnucci ◽

Giulia Amicarelli ◽

Cinzia Pultrone ◽

Veronica Tettamanzi ◽

...

Keyword(s):

Cell Lines ◽

Molecular Detection ◽

Lymphoblastic Leukemia ◽

Internal Quality ◽

Clinical Samples ◽

Wild Type ◽

Rt Pcr ◽

Automatic Instrument ◽

Fusion Transcripts ◽

One Step

Abstract Abstract 2515 Background: The molecular identification of the BCRABL transcripts in clinical samples is actually based on a conventional Reverse Transcription Polymerase Chain reaction (RT-PCR). Here we present a novel molecular method, based on Loop Mediated Amplification assay that, starting from RNA (RT-LAMP) in one single tube reaction ensures a rapid and simultaneous detection of either the BCR-ABL p190 or p210 fusion transcript as well as the housekeeping gene used as internal quality control. Methods: The BCR-ABL RT-LAMP is a multiplex, isothermal method for retro-transcription, amplification and detection of the Minor (p190) and Major (p210) t(9;22) transcripts and the endogenous Gusb RNA, as internal control for validation of negative results. The employment of fluorescent specific probes allows real-time monitoring of the reaction, so that the test result is obtained in a single, homogeneous step. RT-LAMP is carried out on the Liaison IAM, a 8-wells manageable instrument suitable for isothermal reactions. Liaison IAM incubates at constant temperature, monitors the fluorescent signals and the data produced can be analyzed, upon connection to up to 6 other instruments, for a throughput of 8 to 48 samples. Thanks to the three channels of fluorescence, it can monitor multiplex assays, providing elaborated final objective results with no need of further data analysis by the operator. Results: The level of sensitivity of the triplex BCR-ABL RT-LAMP has been analytically evaluated directly on serial dilutions of RNA extracted respectively from t(9;22) positive cell lines (TOM-1 for p190 or K-562 for p210) into wild type RNA from HL-60 cell line (30 replicates). The p190 and p210 transcripts have been detected and distinguished down to 10−4 and 10−5respectively within 50 minutes. The assay demonstrated 100% specificity since 70 replicates of wild type RNA from 7 cell lines resulted BCR-ABL negative and GUSb positive (internal amplification control). This assay was validated on 60 clinical samples (30 white blood cells RNA from Chronic Myeloid Leukemia, 30 mononuclear cells RNA from B-lineage Acute Lymphoblastic Leukemia). All these samples were obtained at diagnosis and were previously analyzed by conventional RT-PCR. RT-LAMP detected and identified the BCR-ABL fusion transcripts correctly in all cases with a 100% concordance with the reference method. Fully concordant results were obtained also on 30 RNA samples from patients affected by t(9;22) negative hematologic malignancies and on 30 RNA samples obtained from healthy donors in which the RT-LAMP amplified exclusively the housekeeping GUSb transcript. Conclusions: The triplex p190-p210-GUSb RT-LAMP is a one-step procedure for specific, highly sensitive and rapid molecular detection of the BCR-ABL fusion transcripts. The semi-automatic instrument Liaison IAM, simplifies the entire procedure, reduces the contamination risks deriving from the conventional, multi step RT-PCR and significantly improve the diagnostic lab routine. Disclosures: D'agostini: DIASORIN S.p.A: Employment. Minnucci:Diasorin S.p.A.: Employment. Amicarelli:DIASPRIN S.p.A.: Employment. Pultrone:DIASORIN S.p.A.: Employment. Tettamanzi:DIASORIN S.p.A.: Employment. Salmoiraghi:DIASORIN S.p.A.: Consultancy. Spinelli:DIASORIN S.p.A: Consultancy. Colotta:DIASORIN S.p.A: Employment. Rambaldi:DIASORIN S.p.A: Consultancy.

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Endogenous H2S production deficiencies lead to impaired renal erythropoietin production

Canadian Urological Association Journal ◽

10.5489/cuaj.5658 ◽

2018 ◽

Vol 13 (7) ◽

Cited By ~ 2

Author(s):

Jennifer Leigh ◽

Smriti Juriasingani ◽

Masoud Akbari ◽

Peng Shao ◽

Manujendra N. Saha ◽

...

Keyword(s):

Cell Lines ◽

Renal Cell ◽

Oxygen Sensor ◽

Whole Body ◽

Clinical Samples ◽

Biosynthetic Enzyme ◽

Wild Type ◽

Renal Cell Lines

Introduction: Patients suffering from chronic kidney disease (CKD) experience a number of associated comorbidities, including anemia. Relative deficiency in renal erythropoietin (EPO) production is thought to be a primary cause of anemia. Interestingly, CKD patients display low levels of hydrogen sulfide (H2S), an endogenously derived renal oxygen sensor. Previous in vitro experiments have revealed that H2S-deficient renal cell lines produce less EPO than wild-type renal cell lines during hypoxia.Methods: We postulated that H2S might be a primary mediator of EPO synthesis during hypoxia, which was tested using an in vivo murine model of whole-body hypoxia and in clinical samples obtained from CKD patients.Results: Following a 72-hour period of hypoxia (11% O2), partial H2S knockout mice (lacking the H2S biosynthetic enzyme cystathionine γ-lyase [CSE]) displayed lower levels of hemoglobin, EPO and cystathionine-β-synthase (CBS) (another H2S biosynthetic enzyme) compared to wild-type mice, all of which was rescued by exogenous H2S supplementation. We also found that anemic CKD patients requiring exogenous EPO exhibited lower urinary thiosulfate levels compared to non-anemic CKD patients of similar CKD classification.Conclusions: Together, our results confirm an interplay between the actions of H2S during hypoxia and EPO production.

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Application of Molecular Docking for the Development of Improved HIV-1 Reverse Transcriptase Inhibitors

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200628103359 ◽

2020 ◽

Vol 16 ◽

Author(s):

Arash Soltani ◽

Seyed Isaac Hashemy ◽

Farnaz Zahedi Avval ◽

Houshang Rafatpanah ◽

Seyed Abdolrahim Rezaee ◽

...

Keyword(s):

Reverse Transcriptase ◽

Binding Capacity ◽

Binding Pocket ◽

Ligand Docking ◽

Binding Modes ◽

Wild Type ◽

Parent Molecule ◽

Discovery Studio ◽

Approved Drugs ◽

Hiv 1

Introoduction: Inhibition of the reverse transcriptase (RT) enzyme of human immunodeficiency virus (HIV) by low molecular weight inhibitors is still an active area of research. Here, protein-ligand interactions and possible binding modes of novel compounds with the HIV-1 RT binding pocket (the wild-type as well as Y181C and K103N mutants) were obtained and discussed. Methods: A molecular fragment-based approach using FDA-approved drugs were followed to design novel chemical derivatives using delavirdine, efavirenz, etravirine and rilpivirine as the scaffolds. The drug-likeliness of the derivatives was evaluated using Swiss-ADME. Then the parent molecule and derivatives were docked into the binding pocket of related crystal structures (PDB ID: 4G1Q, 1IKW, 1KLM and 3MEC). Genetic Optimization for Ligand Docking (GOLD) Suite 5.2.2 software was used for docking and the results analyzed in the Discovery Studio Visualizer 4. A derivative was chosen for further analysis, if it passed drug-likeliness and the docked energy was more favorable than that of its parent molecule. Out of the fifty-seven derivatives, forty-eight failed in druglikeness screening by Swiss-ADME or in docking stage. Results: The final results showed that the selected compounds had higher predicted binding affinities than their parent scaffolds in both wild-type and the mutants. Binding energy improvement was higher for the structures designed based on second-generation NNRTIs (etravirine and rilpivirine) than the first-generation NNRTIs (delavirdine and efavirenz). For example, while the docked energy for rilpivirine was -51 KJ/mol, it was improved for its derivatives RPV01 and RPV15 up to -58.3 and -54.5 KJ/mol, respectively. Conclusion: In this study, we have identified and proposed some novel molecules with improved binding capacity for HIV RT using fragment-based approach.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

International Journal of Molecular Sciences ◽

10.3390/ijms22126565 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6565

Author(s):

Jennifer H. Foster ◽

Eveline Barbieri ◽

Linna Zhang ◽

Kathleen A. Scorsone ◽

Myrthala Moreno-Smith ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Wild Type ◽

Tumor Weight ◽

Cycle Arrest ◽

P53 Status ◽

Neuroblastoma Cell Lines ◽

Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

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Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines

Biochemical Journal ◽

10.1042/bcj20180435 ◽

2019 ◽

Vol 476 (4) ◽

pp. 629-643 ◽

Cited By ~ 1

Author(s):

Isabelle Gerin ◽

Marina Bury ◽

Francesca Baldin ◽

Julie Graff ◽

Emile Van Schaftingen ◽

...

Keyword(s):

Dna Damage ◽

Cell Lines ◽

Oxidative Dna Damage ◽

Fold Increase ◽

Metabolic Effects ◽

Phosphoglycerate Mutase ◽

Wild Type ◽

Metabolic Disturbances ◽

Phosphoglycolate Phosphatase ◽

Damage Repair

Abstract Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4185 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4185-4189 ◽

Cited By ~ 7

Author(s):

J A Greenspan ◽

F M Xu ◽

R L Davidson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Dna Sequences ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Shuttle Vector ◽

Ethyl Methanesulfonate ◽

Wild Type ◽

Single Base ◽

Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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