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Targeted silencing of TRPM7 ion channel induces replicative senescence and produces enhanced cytotoxicity with gemcitabine in pancreatic adenocarcinoma.

ABSTRACT: The transient receptor potential TRPM7 ion channel is required for cellular proliferation in pancreatic epithelia and adenocarcinoma. To elucidate the mechanism that mediates the function of TRPM7, we examined its role in survival of pancreatic cancer cells. RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. TRPM7-deficient cells underwent replicative senescence with up-regulation of p16(CDKN2A) and WRN mRNA. The combination of anti-TRPM7 siRNA and gemcitabine produced enhanced cytotoxicity as compared to gemcitabine alone. Thus, TRPM7 is required for preventing senescence, and modulation of TRPM7 expression may help improve treatment response of pancreatic cancer by combining with apoptosis-inducing agents.

SUBMITTER: Yee NS

PROVIDER: S-EPMC3303226 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Targeted silencing of TRPM7 ion channel induces replicative senescence and produces enhanced cytotoxicity with gemcitabine in pancreatic adenocarcinoma.

Yee Nelson S NS Zhou Weiqiang W Lee Minsun M Yee Rosemary K RK

Cancer letters 20111211 1

The transient receptor potential TRPM7 ion channel is required for cellular proliferation in pancreatic epithelia and adenocarcinoma. To elucidate the mechanism that mediates the function of TRPM7, we examined its role in survival of pancreatic cancer cells. RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. TRPM7-deficient cells underwent replicative senescence with up-regulation of p16(CDKN2A) and WRN mRNA. The combination of anti-TRPM7 siRNA and gemcitabine prod ...[more]

PMID: 22166235

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CTCF Mediates Replicative Senescence Through POLD1.

Project description:POLD1, the catalytic subunit of DNA polymerase δ, plays a critical role in DNA synthesis and DNA repair processes. Moreover, POLD1 is downregulated in replicative senescence to mediate aging. In any case, the components of age-related downregulation of POLD1 expression have not been fully explained. In this article, we elucidate the mechanism of the regulation of POLD1 at the transcription level and found that the transcription factor CCCTC-binding factor (CTCF) was bound to the POLD1 promoter area in two sites. The binding level of CTCF for the POLD1 promoter appeared to be related to aging and was confirmed to be positively controlled by the CTCF level. Additionally, cell senescence characteristics were detected within the cells transfected with short hairpin RNA (shRNA)-CTCF, pLenti-CMV-CTCF, shRNA-POLD1, and pLenti-CMV-POLD1, and the results showed that the CTCF may contribute to the altered expression of POLD1 in aging. In conclusion, the binding level of CTCF for the POLD1 promoter intervened by an age-related decrease in CTCF and downregulated the POLD1 expression in aging. Moreover, the decrease in CTCF-mediated POLD1 transcription accelerates the progression of cell aging.

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Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling.

Project description:The thymic development of regulatory T (Treg) cells, crucial suppressors of the responses of effector T (Teff) cells, is governed by the transcription factor FOXP3. Despite the clinical importance of Treg cells, there is a dearth of druggable molecular targets capable of increasing their numbers in vivo. We found that inhibiting the function of the TRPM7 chanzyme (ion channel and enzyme) potentiated the thymic development of Treg cells in mice and led to a substantially higher frequency of functional Treg cells in the periphery. In addition, TRPM7-deficient mice were resistant to T cell-driven hepatitis. Deletion of Trpm7 and inhibition of TRPM7 channel activity by the FDA-approved drug FTY720 increased the sensitivity of T cells to the cytokine interleukin-2 (IL-2) through a positive feed-forward loop involving increased expression of the IL-2 receptor α-subunit and activation of the transcriptional regulator STAT5. Enhanced IL-2 signaling increased the expression of Foxp3 in thymocytes and promoted thymic Treg (tTreg) cell development. Thus, these data indicate that inhibiting TRPM7 activity increases Treg cell numbers, suggesting that it may be a therapeutic target to promote immune tolerance.

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Effect of truncation on TRPM7 channel activity.

Project description:Transient receptor potential melastatin-like 7 (TRPM7) is a key player in various physiological and pathological processes. TRPM7 channel activity is regulated by different factors. The effects of cleavage of different domains on channel activity remain unknown. Here, we constructed several TRPM7 clones and explored the effects of truncating the mouse TRPM7 at different locations on the ion channel activity in two cell lines. We compared the clones' activity with the full-length TRPM7 and the native TRPM7 in transfected and untransfected cells. We also expressed fluorescently tagged truncated clones to examine their protein stability and membrane targeting. We found that truncating the kinase domain induced reduction in TRPM7 channel activity. Further truncations beyond the kinase (serine/threonine rich domain and/or coiled-coil domain) did not result in further reductions in channel activity. Two truncated clones lacking the TRP domain or the melastatin homology domain had a completely nonfunctional channel apparently due to disruption of protein stability. We identified the shortest structure of TRPM7 with measurable channel activity. We found that the truncated TRPM7 containing only S5 and S6 domains retained some channel activity. Adding the TRP domain to the S5-S6 resulted in a significant increase in channel activity. Finally, our analysis showed that TRPM7 outward currents are more sensitive to truncations than inward currents. Our data provide insights on the effects of truncating TRPM7 at different locations on the channel functions, highlighting the importance of different domains in impacting channel activity, protein stability, and/or membrane targeting.

| S-EPMC10761173 | biostudies-literature

Expression profiles from HEK293 cells by overexpression of TRPM7 channel

Project description:The functional activity of TRPM7 is essential for cell viability and growth, and its expression is up-regulated in certain pathological conditions. In order to assess the effects of TRPM7 activity on cellular gene expression, inducible HEK293 cell-lines harboring the wild-type mouse TRPM7 and a mutant lacking the kinase domain were established. By using microarray analysis, we identified genetic profiles altered in transcription significantly and specifically by the expression of the functional TRPM7 channel. Overexpression of TRPM7 channel in HEK cells induce the cell damage and cell death. So we used tetracycline-inducible system for channel expression. However, since the treatment of inducer itself could also affect the transcription profile, we established another cell-line harboring a nonfunctional TRPM7 mutant (TRPM7DKD) as a control in which the entire kinase domain at the C-terminus was deleted. The wild-type and the non-functional TRPM7 channels were induced transiently, and the comparative changes in cellular transcription were investigated

2011-07-22 | E-GEOD-23102 | biostudies-arrayexpress

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