Recombinant Lentivirus-Mediated Silencing of Adaptor Protein Ruk/Cin85 Expression Influences Biological Responses of Tumor Cells

Samoylenko, Anatoly; Byts, Nadiya; Pasichnyk, G. V.; Kozlova, Nina; Bazalii, A. V.; Gerashchenko, D. S.; Shandrenko, S. G.; Vorotnikov, Alexander V.; Kietzmann, Thomas; Komisarenko, Serhiy; Дробот, Л. Б.

doi:10.15407/biotech6.04.182

Cited by 4 publications

(7 citation statements)

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“…According to our previous results, the adaptor protein Ruk/CIN85 can form the intracellular doi: http://dx.doi.org/10.15407/ubj88.01.119 complex with adaptor protein Tks4 [13,14], which functions as organizer subunit of NADPH-oxidase complex mediated by Nox1 [15,16]. We have also revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with adaptor protein Ruk/CIN85 expression [17]. Using expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with adaptor protein Ruk/CIN85 and live cell fluorescence microscopy, the co-localization of Ruk/CIN85 and Н 2 О 2 generation was shown to take place at the edges of representative vesicular structures in transiently transfected MCF-7 cells [18].…”

mentioning

confidence: 51%

“…To study the possible influence of adaptor protein Ruk/CIN85 on transcriptional regulation of NoX genes, human breast adenocarcinoma MCF-7 cell line and its sublines with different levels of adaptor protein Ruk/CIN85 overexpression (D4<G10<G4) [18] as well as G4 cells, stably infected with lentivirus encoding Ruk/CIN85-specific shRNA (G4shRuk) [17], were used. The expression levels of NoX genes 1-5, DUoX genes 1-2 and gene for accessory protein p22 Phox were evaluated by qPCR relative to housekeeping gene yWHAz.…”

Section: Resultsmentioning

confidence: 99%

“…In cell lines of epithelial origins, high levels of NoX1 mRNA expression were observed in colorectal cell lines [8,23], especially in HT-29 cells, for which the positive correlation between ROS production and expression of Ruk/CIN85 was demonstrated by us [17]. High levels of NoX1, NoX2, DUoX1 and DUoX2 genes expression were also detected in lung adenocarcinoma A549 cells, and ROS production by these cells was partially reduced by NADPH oxidase inhibitor diphenyleniodonium [24].…”

Section: Primer Sequences For Rt-pcr Amplificationmentioning

confidence: 99%

“…cell culture. Cell line MCF-7 (human breast adenocarcinoma) and its sublines with Ruk/CIN85 overexpression D4, G4 and G10 [19], and G4 cells stably infected with lentivirus encoding Ruk/ CIN85-specific shRNA [17] were maintained in DMEM (Gibco ® ) containing 10% fetal bovine serum (HyСlone), 2 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco ® ) at 37 °C and 5% CO 2 in a humidified atmosphere. Cells were split 1:3-1:5 every 2-3 days at 70-80% confluency.…”

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confidence: 99%

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Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

Bazalii¹,

Horak²,

Pasichnyk³

et al. 2016

Ukr.Biochem.J.

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NADPH oxidases are key components of redox-dependent signaling networks involved in the control of cancer cell proliferation, survival and invasion. The data have been accumulated that demonstrate specific expression patterns and levels of NADPH oxidase homologues (NOXs) and accessory genes in human cancer cell lines and primary tumors as well as modulation of these parameters by extracellular cues. Our previous studies revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with adaptor protein Ruk/CIN85 expression while increased levels of Ruk/CIN85 in weakly invasive human breast adenocarcinoma MC F-7 cells contribute to their malignant phenotype through the constitutive activation of Src/Akt pathway. In this study, to investigate whether overexpression of Ruk/CIN85 in MC F-7 cells can influence transcriptional regulation of NOXs genes, the subclones of MCF-7 cells with different levels of Ruk/CIN85 were screened for NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 as well as for regulatory subunit p22Phox mRNA contents by quantitative RT-PCR (qPCR). Systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control WT cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes.

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confidence: 51%

Section: Resultsmentioning

confidence: 99%

Section: Primer Sequences For Rt-pcr Amplificationmentioning

confidence: 99%

mentioning

confidence: 99%

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Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

Bazalii¹,

Horak²,

Pasichnyk³

et al. 2016

Ukr.Biochem.J.

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“…Ruk/CIN85overexpressing subline RukUp and corresponding control subline Mock were obtained by Ca-phosphate transfection of 4T1 cells with pRc/CMV2-Rukl or empty vector, respectively [23]. Ruk/CIN85 expression was suppressed in subline called RukDown using lentiviral construction pLKO.1-shRuk/CIN85 R22 [24] and for generation of control subline (Scr) nontargeting vector was used. Transfected/infected cells were subcloned and selected with 1mg/ml geneticin (G418) or 10 µg/ml puromycin, respectively.…”

Section: Methodsmentioning

confidence: 99%

Adaptor protein Ruk/CIN85 modulates resistance to doxorubicin of murine 4T1 breast cancer cells

Horak¹,

Gerashchenko²,

Drobot³

2018

Ukr.Biochem.J

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the acquisition of chemoresistance in the course of tumor progression includes activation of membrane ABC transporters, detoxification enzymes, cell cycle deceleration and activation of specific signaling pathways such as Akt/mTOR, MAPK, NF-κB. Adaptor proteins play an essential role in the assembly of supramolecular signaling complexes, maintaining and directing the intracellular signaling. one of such proteins, called Ruk/cIN85, is strongly associated with malignant transformation and metastasis. In present study we investigated the Ruk/CIN85 effect of up/down-regulation on the transforming potential and doxorubicin resistance of highly aggressive mouse breast adenocarcinoma 4t1 cells. It was demonstrated that 4t1 cells overexpressing Ruk/cIN85 possessed increased resistance to doxorubicin (in the range of concentrations 0.1-10.0 µM) while knockdown cells were the most sensitive. also, high levels of Ruk/cIN85 in 4t1 cells positively correlated with their ability to form colonies in semi-solid agar. Ruk/cIN85-overexpressing cells formed four times more colonies in comparison with Ruk/cIN85 nockdown cells, the growth of which revealed higher resistance to doxorubicin action. k e y w o r d s: Breast cancer, chemoresistance, adaptor proteins, Ruk/cIN85.

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Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

2016

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To study a possible involvement of NADPH oxidases in the control of cell motility and Akt signaling in the human breast adenocarcinoma MCF-7 cells that stably overexpress the full-length form of adaptor protein Ruk/CIN85. Methods. Cell motility was studied by a transwell migration assay. The dynamics of EGFinduced Akt activation was investigated by Western blot analysis. Results. It has been shown that apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidases, substantially attenuates the motility of Ruk/CIN85 overexpressing MCF-7 cells (subclone G10) in comparison with the control cells. In addition, apocynin induced the transition from sustained to transient EGF-dependent Akt activation in G10 cells and did not influence transient Akt activation in the control cells. Conclusions. Data obtained can suggests that ROS produced by NADPH oxidases are signaling components, upstream to Akt kinase, that mediate the increased migratory potential of Ruk/CIN85-overexpressing MCF-7 cells.

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Recombinant Lentivirus-Mediated Silencing of Adaptor Protein Ruk/Cin85 Expression Influences Biological Responses of Tumor Cells

Cited by 4 publications

References 12 publications

Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

Adaptor protein Ruk/CIN85 modulates resistance to doxorubicin of murine 4T1 breast cancer cells

Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

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