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Background: Renal T cells contribute importantly to hypertension, but the underlying mechanism is incompletely understood. We reported that CD8Ts directly stimulate distal convoluted tubule cells (DCTs) to increase NCC (sodium chloride co-transporter) expression and salt reabsorption. However, the mechanistic basis of this pathogenic pathway that promotes hypertension remains to be elucidated. Methods: We used mouse models of DOCA+salt (DOCA) treatment and adoptive transfer of CD8 + T cells (CD8T) from hypertensive animals to normotensive animals in in vivo studies. Co-culture of mouse DCTs and CD8Ts was used as in vitro model to test the effect of CD8T activation in promoting NCC-mediated sodium retention and to identify critical molecular players contributing to the CD8T-DCT interaction. Interferon (IFNγ)-KO mice and mice receiving renal tubule-specific knockdown of PDL1 were used to verify in vitro findings. Blood pressure was continuously monitored via radio-biotelemetry, and kidney samples were saved at experimental end points for analysis. Results: We identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFN-γ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFN-γ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in 2 different mouse models. Conclusions: Our results identified the role of activated CD8Ts in contributing to increased sodium retention in DCTS through the IFNγ-PDL1 pathway. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions. NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heart failure is the top killer in the U.S and worldwide, often caused by hypertension, aging, or cardiac toxicity. Diastolic Dysfunction (DD), the often the first step of heart failure, occurs when the chambers of the heart are not able to expand during the filling phase. In addition, hypertension is the leading cause of DD. Though this is known, the progression of hypertension into DD remains poorly understood. Of note is the ability of M2 macrophages to differentiate into a myofibroblast-like phenotype, known as macrophage to myofibroblast transition (MMT). This transition has been observed in interstitial fibrosis in chronic renal allograft injury. The researchers noted that MMT account for 50% of the myofibroblast population. To our knowledge, the extent of fibrotic remodeling due to MMT in the heart has not been ardently investigated. Therefore, we aim to answer whether MMT plays a causal role in the progression of hypertension to DD and the pathogenesis of heart failure.Utilizing the uninephrectomized, DOCA-salt model, mice were subjected to this treatment and hypertension was induced. As a negative control, another group of mice received a uninephrectomy and the same salt in their drinking water, however, DOCA was not administered (Sham group). MMT prevalence, identified as CD68+ α-SMA+ expressions, was observed inside the hearts of DOCA and Sham mice. The hearts were isolated and homogenized via gentleMACS tissue Dissociator, the monocytes were purified via Percoll isolation, and flow cytometry was used to identify the population of CD68+ α-SMA+ expressing cells.As soon as 10 days post DOCA implantation, the DOCA group exhibited worsening diastolic function as it took longer for the pressure to change in the hearts of DOCA mice than in Sham (-dp/dt). These data were gathered via left ventricular (LV) catheter insertion. At a later time, utilizing a different cohort of mice preliminary data show an increase of monocyte (CD68+) and fibroblast (α-SMA+) double-positive expressing cells in the hearts of hypertensive mice.MMT has been observed in the hearts of hypertensive mice. This transition may exacerbate Diastolic Dysfunction. More research must be performed to investigate the wide variety of functions of macrophages (i.e., M1 to M2 ratio and transition, macrophage residence or infiltration, and macrophage stimulation of myofibroblasts), as well as the specific interactions between macrophages and cardiomyocytes, or other immune cells, that might mediate this transition. These data provide a preliminary, but interesting glimpse, into the role of macrophages in exacerbating Diastolic Dysfunction and mediating Heart Failure. NIH RO1-HL146713 and AHA 15BGIA25730047 (Shengyu Mu) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background and Hypothesis: T cells play a critical role in hypertension; however, the molecules driving this interaction were unclear. We recently showed that CD8Ts are activated during the initial phase of blood pressure elevation and directly interact with the distal convoluted tubule (DCT) through the interferon gamma (IFNγ)-PDL1 pathway. Here we identified that these CD8Ts are activated during this initial phase via pro-inflammatory extracellular ATP stimulating their P2X7 receptors, leading to enhanced IFNγ production and increased sodium retention in tubular cells that interacted with these CD8Ts. We predict the CD8T P2X7 receptor is contributing to hypertension and abrogating this receptor on CD8Ts will block CD8T-tubular interaction and lower blood pressure. Methods: To determine whether CD8Ts activation in hypertension requires T cell receptor (TCR) stimulation, we used transgenic OT1 mice and induced hypertension via the DOCA + salt (DOCA) model, measuring blood pressure and confirming enhanced CD8T activation despite no exposure to ovalbumin. In in-vitro studies, we co-cultured DCTs with pre-stimulated CD8Ts and measured PDL1 expression and sodium retention in DCTs via flow cytometry. CD8Ts were obtained from WT or P2X7 KO mice and stimulated with BZATP or ATP using calcium influx (intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (Seahorse) as activation endpoints. To confirm in-vivo, the activation profiles of CD8Ts obtained from sham or DOCA treated WT and P2X7 KO mice were measured using rt-qPCR and flow cytometry. To confirm the role of P2X7 receptors on CD8Ts, we performed adoptive transfer of DOCA CD8Ts (WT or P2X7 KO) to the alternative model and transferred naive WT CD8Ts to P2X7 KO mice receiving DOCA treatment. All blood pressure was measured via radio-biotelemetry. Results: We found OT1 CD8Ts become activated to the DOCA presser, produce more IFNγ, and contribute to salt-sensitive hypertension without antigen-mediated TCR stimulation. P2X7 receptor agonists BZATP and ATP drove CD8T calcium influx, increased IFNγ transcription, and increased glycolytic activity in CD8Ts – indicative of T cell activation; as such, CD8Ts pre-stimulated with the P2X7 agonist BZATP increased tubular PDL1 expression and sodium retention more than naive CD8Ts in our co-culture model. DOCA treated P2X7 KO CD8Ts were unable to increase blood pressure in our adoptive transfer model; however, transferring sham WT CD8Ts to DOCA P2X7 KO mice restored blood pressure response to the presser to WT levels. Conclusion: Stimulation of the P2X7 receptor contributes to CD8T activation in the pathogenesis of hypertension, resulting in enhanced sodium retention. Deficiency of this receptor on CD8Ts prevents activation by ATP and results in blunted blood pressure elevation in two models of hypertension; as such, the CD8T P2X7 receptor may be a promising target for improving blood pressure management. NIH R01-HL146713, AHA 15BGIA25730047, SPaT 5T32GM106999-09 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypertension is the leading cause of cardiovascular disease and the primary risk factor for mortality worldwide. For more than half a century, researchers have demonstrated that immunity plays an important role in the development of hypertension; however, the precise mechanisms are still under investigation. The current body of knowledge indicates that proinflammatory cytokines may play an important role in contributing to immune-related pathogenesis of hypertension. Interferon gamma (IFN-γ), in particular, as an important cytokine that modulates immune responses, has been recently identified as a critical regulator of blood pressure by several groups, including us. In this review, we focus on exploring the role of IFN-γ in contributing to the pathogenesis of hypertension, outlining the various immune producers of this cytokine and described signaling mechanisms involved. We demonstrate a key role for IFN-γ in hypertension through global knockout studies and related downstream signaling pathways that IFN-γ production from CD8(+) T cell (CD8T) in the kidney promoting CD8T-stimulated salt retention via renal tubule cells, thereby exacerbating hypertension. We discuss potential activators of these T cells described by the current literature and relay a novel hypothesis for activation.

Background & Hypothesis: The immune system plays a critical role in the development of hypertension. We recently found that CD8 + T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), upregulating sodium-chloride co-transporter (NCC) activity, leading to excessive sodium retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that activated CD8Ts release gamma interferon (IFNγ) that primes the tubular cell to express PDL1 which functions as a co-stimulatory ligand promoting interactions between activated CD8Ts and DCTs contributing to the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice exhibit higher activity compared to those from sham normotensive mice. Pre-activated CD8Ts demonstrated augmented ability to interact with DCTs compared to naïve CD8Ts, and as a consequence, DCTs that had been co-cultured with pre-activated CD8Ts demonstrated increased expression of NCC (7 fold) and increased PDL1 and high sodium retention (47% vs. 0-3%). These effects were abolished by neutralizing IFNγ or knockdown of PDL1 in DCTs. In-vivo results verified the in-vitro studies. We found that IFNγ-KO mice demonstrated reduced blood pressure elevation and T cell infiltration within their kidneys to DOCA-salt compared to WT mice receiving the same treatment (last day systolic blood pressure average 155.2 mmHg compared to 189 mmHg from WT). Blood pressure elevation was blunted in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1 (last day systolic blood pressure average 124 mmHg for mice with siPDL1 nanoparticle and 144 mmHg for mice with scrambled siRNA); NCC elevation was also blunted in this model. Conclusion: Activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNγ-PDL1 mediated mechanism. Blocking the IFNγ-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo , thereby ameliorating salt-sensitive hypertension.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01-3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT: ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

In recent years, robotic training has been utilized for recovery of motor control in patients with motor deficits. Along with clinical assessment, electrical patterns in the brain have emerged as a...

As a promising cancer treatment modality that has emerged, photothermal therapy can harness antitumor immunity by triggering immunogenic cell death (ICD) in addition to direct cell ablation. However, the immuno-stimulation induced by PTT alone is insufficient to achieve satisfactory cancer eradication, especially in immunologically "cold" tumors due to their harsh immunosuppressive microenvironment. Effective activation of the innate immune system is indispensable to boost a robust adaptive antitumor immune response typically initiated by dendritic cells (DCs). Herein the above issues are addressed by constructing an environmentally responsive supramolecular nanoself-assembly (PSAs) derived from a novel polypeptide-based block copolymer, which is capable of co-load photothermal immunomodulators efficiently under structure-guided π-π stacking interactions. In the murine model of 4T1 xenograft tumors, the fabricated PSAs with payloads trigger both adaptive and innate immune responses in situ through activation of ICD as well as STING-dependent signal pathway. The findings reveal a new mechanism of harnessing photothermal therapy toward immunologically "cold" tumors.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01–10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8+ T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8+ T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8+ T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K+ channel Kir4.1, and stimulation of the Cl− channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension., T cells contribute to development of high blood pressure but their role in salt-sensitive hypertension is less clear. Liu et al. show that CD8+ T cells upregulate and activate Na-Cl co-transporter NCC in distal convoluted tubules via direct cell-cell contact and ROS-Src activation, leading to Na+ retention and salt-sensitive hypertension.

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention is an important question, which if answered, could reveal a critical relationship between adaptive immunity and pathogenesis of salt-sensitive hypertension. In the present study, we propose a novel mechanism of salt-sensitive hypertension via the distal nephron: that in salt-sensitive hypertension, renal infiltrating T cells interact with distal convoluted tubule cells (mDCTs), which leads to up-regulation of the sodium-chloride-co-transporter (NCC), and thus enhancement of sodium retention. In vivo tests used DOCA-salt mice (SBP~190mmHg on a high salt diet) and T cell-adoptive transferred mice (SBP~170mmHg on a high salt diet). Results confirmed enhanced NCC & p-NCC expression (>2.5X) in the kidneys of both mouse models. We found significantly more T cells infiltrating the kidneys of DOCA-salt & T cell-transferred mice compared to their controls. It is noteworthy that in both models a great number of CD8 + T cells (CD8Ts) in the renal tubulointerstitium surround DCT segments which have higher levels of NCC expression. Direct contact between these two cell types was confirmed by super-resolution structured illumination microscopy. In vitro, we co-cultured mDCTs with T cells, and found that CD8Ts, but not CD4Ts, significantly increased NCC expression (>3X) in mDCTs via “direct cell-cell contact”. An intracellular sodium-indicator detected higher NCC-mediated sodium uptake (>1.5X) in mDCTs treated with CD8Ts compared to untreated controls. Moreover, these effects were mediated by increased (>2.2X) functional chloride channel Clc-k in the cell membrane. Clc-k knockdown by siRNA blunted chloride efflux and abolished CD8T-induced NCC activation and sodium retention in mDCTs. Taken together, our findings suggest a novel role for CD8 + T cells in enhancement of salt retention contributing to salt-sensitive hypertension: that CD8 + T cells directly contact mDCTs, stimulate NCC by upregulating the chloride channel Clc-k on the mDCT plasma membrane, thereby increasing chloride efflux, which leads to compensatory chloride influx via NCC activation at the cost of increasing sodium influx.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived arachidonic acid metabolite endowed with vasoconstrictor. Recent reports documented that 20-HETE synthesis inhibitors decrease the growth of glioma and renal cell carcimona, and that urinary 20-HETE levels are significantly elevated in prostate cancer patients. These reports led us to investigate the role of 20-HETE in the regulation of blood flow to prostate cancer tumors. First, we compared 20-HETE levels in prostate cancer cell line-LNCaP and normal prostate epithelial cells. Cultured LNCaP cells produced high levels of 20-HETE (566±139 pg/mg protein), whereas 20-HETE production was undetectable in normal prostate epithelial cells. Second, subcutaneous tumors were induced in balb/c nude mice by injection of LNCaP cells, followed by assessment of 20-HETE in both tumors and isolated tumor vessels. We found that LNCaP cell-derived tumors had high levels of 20-HETE (425±97 pg/mg protein), while the level in isolated tumor vessels was undetectable. Third, we used laser-doppler flowmetry to evaluate whether tumor-derived 20-HETE influences tumor blood flow and vessel diameter in vivo. Topical application of the 20-HETE antagonist 20-6,15-HEDE (1 mM) to tumor vessels in vivo increased blood flow by 1.81±0.34-fold and vessel diameter by 1.286±0.133-fold, which responses were reversed by topical application of 20-HETE in a dose-dependent manner. We conclude that LNCaP cell-derived tumors produce 20-HETE which, in turn, promotes vasoconstriction leading to reduction of tumor blood flow.

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP450-derived arachidonic acid metabolite in the microcirculation, contributes to vascular tone and stimulates vascular and epithelial cell proliferation, angiogenesis and inflammation, all of which play a role in cancer progression. Our laboratory identified 20-HETE as the mediator of androgen-induced hypertension. Given that androgen contributes to the pathogenesis of prostate cancer, we examined the presence and function of 20-HETE in human prostate cancer cells. Androgen-responsive prostate cancer cells, LNCaP, produced 2-fold more 20-HETE compared to androgen-nonresponsive C4-2 cells (565±139 pg/mg protein vs 223±50 pg/mg protein). Both C4-2 and LNCaP produced greater amount of 20-HETE compared with U87 glioma, MCF-7 breast cancer and ML-1 thyroid cancer cells. 20-HETE levels in prostate primary stromal cells and epithelial RWPE-1 cells were barely detected (16±26 and 23±40 pg/mg protein respectively). Treatment of LNCaP with 5 alpha-dihydrotestosterone (DHT) stimulated 20-HETE production by 65% when compared to vehicle control. DHT treatment did not alter 20-HETE levels in C4-2. Treatment of C4-2 and LNCaP with HET0016, an inhibitor of 20-HETE synthesis, selectively decreased endogenous 20-HETE production while inhibiting cell growth in a dose-dependent manner. Further addition of exogenous 20-HETE stimulated growth. CYP4F2, CYP4F3 and CYP4F8 mRNA levels were several fold higher in LNCaP compared with C4-2, while levels of CYP4A11 and CYP4A22 mRNA were unchanged. In LNCaP, DHT increased CYP4F isoform expression without altering the CYP4A expression; flutamide abrogated DHT-induced expression of CYP4F2 and CYP4F3 but did not affect expression of CYP4F8. Importantly, flutamide inhibited DHT-stimulated 20-HETE production. These results suggest that 20-HETE has an important role in cancer cell proliferation and CYP4F2/3 might be the source of androgen-driven 20-HETE synthesis via androgen receptor dependent pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-160. doi:1538-7445.AM2012-LB-160

Background The trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo. Methodology and Principal Findings The molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E.coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacIQ in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA. Conclusions and Significance Our findings thus suggest that the longer AAT•ATT triplet repeats in E.coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.