Chemoradiation Improves Survival Compared With Chemotherapy Alone in Unresected Nonmetastatic Gastric Cancer.

Background: Limited data are available to guide management of patients with stage I-III gastric cancer not undergoing potentially curative surgical resection. We compared survival outcomes associated with chemotherapy alone versus chemoradiation (CRT) in the treatment of nonmetastatic gastric cancer. Methods: Patients with gastric adenocarcinoma from 2004 to 2015 were identified using the National Cancer Database. Patients were excluded if they had surgery, metastatic disease, or T0, Tis, or T1a disease. Logistic regression was used to evaluate predictors of CRT use. Cox proportional hazards modeling was performed to compare overall survival (OS) between chemotherapy alone and CRT in overall and propensity score-matched cohorts. Results: We identified 4,795 patients with stage I-III gastric adenocarcinoma who did not undergo surgery, at a median follow-up of 11.8 months. A total of 3,316 patients (69.2%) received chemotherapy alone and 1,479 patients (30.8%) received CRT. Predictors of increased CRT use were age ≥65 years (odds ratio [OR] 1.68; 95% CI, 1.43-1.99; P<.001), Charlson-Deyo comorbidity score ≥2 (OR, 1.46; 95% CI, 1.18-1.81), and treatment at a community facility (OR, 1.27; 95% CI, 1.07-1.51; P=.006). Patients receiving CRT had a 2-year OS rate of 28.3% compared with 21.5% among those receiving chemotherapy. Multivariate analysis showed that CRT was associated with improved OS (hazard ratio [HR], 0.82; 95% CI, 0.77-0.89; P<.001). After propensity score matching, a persistent survival benefit was observed (HR, 0.80; 95% CI, 0.74-0.88; P<.001). Conclusions: In patients with stage I-III gastric cancer not undergoing surgical resection, CRT was associated with improved survival compared with chemotherapy alone. However, only 30.8% of patients received CRT in this setting.

Surgery Versus Radiation Treatment for High-Grade Neuroendocrine Cancer of Uterine Cervix: A Surveillance Epidemiology and End Results Database Analysis.

OBJECTIVES: The objective of this study was to assess treatment and other factors impacting survival in cervical high-grade neuroendocrine carcinoma (HGNEC). METHODS/MATERIALS: We identified patients with cervical HGNECs diagnosed during 1988 to 2012 in the Surveillance Epidemiology and End Results database. We determined overall survival by International Federation of Gynecology and Obstetrics stages and by local treatment modalities, that is, radical surgery versus external beam radiation treatment (EBRT) plus brachytherapy using Kaplan-Meier analysis with log-rank test. We also determined factors of age, stage, and treatment modality impacting survival using proportional hazard analysis. RESULTS: We identified 832 cases of cervical HGNECs in the database. After excluding cases with incomplete stage data, the International Federation of Gynecology and Obstetrics stages I to IV distributions were 196 (28.0%), 69 (9.9%), 175 (25.0%), and 260 patients (37.1%), respectively. Radical surgery and primary radiotherapy yielded similar 5-year overall survival for stages I (61% vs 53%, P = 0.27), II (48% vs 28%, P = 0.308), and III (33% vs 28%, P = 0.408) patients. External beam radiation treatment plus brachytherapy did not yield superior survival than EBRT alone in stage I (48% vs 49%, P = 0.799), II (37% vs 20%, P = 0.112), or III (25% vs 32%, P = 0.636) patients. Age (P = 0.004) and stage (stage II: hazard ratio [HR], 1.78, P = 0.013; stage III: HR, 2.42; P < 0.001) were independent factors impacting survival but not local treatment modality (EBRT: HR, 1.30, P = 0.17; EBRT plus brachytherapy: HR, 1.16; P = 0.417). CONCLUSIONS: Patients with cervical HGNECs had poor prognosis. Primary treatment by radical surgery or external beam radiotherapy with or without brachytherapy yielded equally poor survival.

DMSO Enhances TGF-ß Activity by Recruiting the Type II TGF-ß Receptor From Intracellular Vesicles to the Plasma Membrane.

Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-ß activity. Here we show that DMSO enhances TGF-ß activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-ß-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-ß receptors (TßR-I and/or TßR-II) by ∼3-4-fold without altering their cellular levels as determined by (125) I-labeled TGF-ß-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TßR-II (but not TßR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TßR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TßR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TßR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TßR-II-HA-containing vesicles from the cytoplasm and co-localization of TßR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-ß activity presumably by inducing fusion of cytoplasmic vesicles (containing TßR-II) and the plasma membrane, resulting in increased localization of TßR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. © 2015 Wiley Periodicals, Inc.

The ratio of weight loss to planning target volume significantly impacts setup errors in nasopharyngeal cancer patients undergoing helical tomotherapy with daily megavoltage computed tomography.

BACKGROUND: Changes in head and neck anatomy during radiation therapy (RT) produce setup uncertainties of nasopharyngeal cancer (NPC) irradiation. We retrospectively analyzed image guidance data to identify clinical predictors of setup errors. PATIENTS AND METHODS: The data of 217 NPC patients undergoing definitive RT on a helical tomotherapy (HT) unit were analyzed. Factors including tumor stage, body mass index, weight loss, and planning target volume (PTV) were assessed as predictors of daily megavoltage computed tomography (MVCT) setup displacements, which were automatically registered using software. RESULTS: Mean daily setup displacements (in mm) were 1.2 ± 0.6, 1.8 ± 0.8, 3.4 ± 1.4 in the medial-lateral (ML), superior-inferior (SI), and anterior-posterior (AP) directions, respectively. Mean weight loss was 4.6 ± 3.3 kg (6.8 ± 4.9%). Patients with weight loss > 5% had significantly larger setup displacements in the AP (3.6 ± 1.5 vs. 2.9 ± 1.1 mm, p < 0.001) and SI (1.6 ± 0.7 vs. 1.9 ± 0.9 mm, p = 0.01) direction, but not in the ML direction (p = 0.279). The AP setup error increased 0.06 mm (y = 0.055x + 2.927, x: percentage of weight loss/PTV, y: AP displacement) per one percent increase in weight loss normalized to PTV. CONCLUSIONS: Patients with weight loss > 5% and smaller PTVs, possibly because of small body frame or neck girth, were more likely to have increased setup errors in the AP direction.

The outcome and prognostic factors for lymph node recurrence after node-sparing definitive external beam radiotherapy for localized prostate cancer.

BACKGROUND: The prognostic factors for the recurrence of lymph node (LN) metastasis after dose-escalated radiotherapy (RT) in prostate cancer patients have not been well investigated. We report the prognostic factors and outcomes in patients receiving salvage treatment for LN recurrence after high-dose intensity-modulated RT (IMRT). METHODS: We studied a cohort of 419 patients with localized prostate adenocarcinoma undergoing definitive IMRT (78 Gy). LN recurrence was diagnosed by size criteria using computed tomography (CT) or magnetic resonance imaging, or abnormal uptake of (18)F-fluorocholine by LNs on positron emission tomography/CT. Overall survival and LN recurrence-free survival (LNRFS) were calculated, and prognostic factors were evaluated. RESULTS: With a median follow-up of 60 months, 18 patients (4.3 %) had LN recurrence and a significantly lower 5-year overall survival rate (60 vs. 90 %, p = 0.003). Univariate analysis showed that T3/T4 stage (p = 0.003), Gleason score >7 (p < 0.001), and estimated risk of pelvic LN involvement of >30 % by the Roach formula (p = 0.029) were associated with significantly lower LNRFS. On multivariate analysis, high Gleason score (hazard ratio = 5.99, p = 0.007) was the only independent factor. The 1/2-year overall survivals after LN recurrence were 67/54 %. Patients with isolated LN recurrence (p = 0.003), prostate-specific antigen (PSA) doubling time >5 months (p = 0.009), interval between PSA nadir and biochemical failure >12 months (p = 0.035), and PSA <10 ng/ml at LN recurrence (p = 0.003) had significantly better survival. Patients with isolated LN recurrence had significantly better survival when treated with combined RT and hormones than when treated with hormones alone (p = 0.011). CONCLUSIONS: Gleason score of >7 may predict LN recurrence in prostate cancer patients treated with definitive IMRT. Small number of patients limits the extrapolation of this risk with the primary treatment strategy. Combined RT and hormones may prolong survival in patients with isolated LN recurrence.

Intra-fractional corrections and clinical outcomes in frameless image-guided radiosurgery for trigeminal neuralgia.

Purpose: Precision targeting is crucial to successful stereotactic radiosurgery for trigeminal neuralgia (TGN). We investigated the impact of intra-fractional 6-dimensional corrections during frameless image-guided radiosurgery (IGRS) for pain outcome in TGN patients. Materials and methods: A total of 41 sets of intra-fractional corrections from 35 patients with TGN treated by frameless IGRS from 2009 to 2013 were retrospectively studied. For each IGRS, the intra-fractional 6-dimensional shifts were conducted at 6 couch angles. Clinical pain outcome was recorded according the Barrow Neurological Institute (BNI) 5-points score. The relationship in 6-dimensional corrections and absolute translational distances between patients with pain relief score points <2 versus ≥2 were analyzed. Results: The absolute mean lateral, longitudinal, and vertical translational shifts were 0.46 ± 0.15 mm, 0.36 ± 0.16 mm and 0.21 ± 0.08 mm, respectively, with 97% of translational shifts being within 0.7 mm. The absolute mean lateral (pitch), longitudinal (roll), and vertical (yaw) rotational corrections are 0.33 ± 0.24°, 0.18 ± 0.09°, and 0.27 ± 0.15°, respectively, with 97% of rotational corrections being within 0.6°. The median follow-up duration for pain outcome was 26 months after IGRS. The average calculated absolute shift for patients with pain relief <2 and ≥2 BNI points, were 0.228 ± 0.008 mm and 0.259 ± 0.007 mm, respectively. There was no statistically significant difference in the translational shifts, rotational corrections or absolute distances between these two patient groups. Conclusions: Our data demonstrate high spatial targeting accuracy of frameless IGRS for TGN with only nominal intra-fraction 6-dimensional corrections.

CRSBP-1/LYVE-1 ligands disrupt lymphatic intercellular adhesion by inducing tyrosine phosphorylation and internalization of VE-cadherin.

Cell-surface retention sequence (CRS) binding protein (CRSBP-1) is a membrane glycoprotein identified by its ability to bind PDGF-BB and VEGF-A via their CRS motifs (clusters of basic amino acid residues). CRSBP-1 is identical to LYVE-1 and exhibits dual ligand (CRS-containing proteins and hyaluronic acid) binding activity, suggesting the importance of CRSBP-1 ligands in lymphatic function. Here, we show that CRSBP-1 ligands induce disruption of VE-cadherin-mediated intercellular adhesion and opening of intercellular junctions in lymphatic endothelial cell (LEC) monolayers as determined by immunofluorescence microscopy and Transwell permeability assay. This occurs by interaction with CRSBP-1 in the CRSBP-1-PDGFßR-ß-catenin complex, resulting in tyrosine phosphorylation of the complex, dissociation of ß-catenin and p120-catenin from VE-cadherin, and internalization of VE-cadherin. Pretreatment of LECs with a PDGFßR kinase inhibitor abolishes ligand-stimulated tyrosine phosphorylation of VE-cadherin, halts the ligand-induced disruption of VE-cadherin intercellular adhesion and blocks the ligand-induced opening of intercellular junctions. These CRSBP-1 ligands also induce opening of lymphatic intercellular junctions that respond to PDGFßR kinase inhibitor in wild-type mice (but not in Crsbp1-null mice) as evidenced by increased transit of injected FITC-dextran and induced edema fluid from the interstitial space into lymphatic vessels. These results disclose a novel mechanism involved in the opening of lymphatic intercellular junctions.

Targeting human epidermal growth factor receptor 2 enhances radiosensitivity and reduces the metastatic potential of Lewis lung carcinoma cells.

BACKGROUND: Sublethal radiation induces matrix metalloproteinase 9 (MMP-9)-mediated radioresistance in Lewis lung carcinoma (LLC) cells and their metastatic dissemination. We aim to determine if EGFR/HER2 activation associates with MMP-9-mediated radioresistance and invasiveness in irradiated LLC cells. METHODS: LLC cells were treated with erlotinib or afatinib followed by sublethal radiation. After irradiation, we examined the phosphorylation of EGFR/HER2 and MMP-9 expression. Colony formation assay determined if the kinase inhibitors sensitize LLC cells to radiation. Matrigel-coated Boyden chamber assay assessed cellular invasiveness. Resulting tumors of wild-type LLC cells or HER2 knock-down mutant cells were irradiated to induce pulmonary metastases. RESULTS: Afatinib more effectively sensitized LLC cells to radiation and decreased invasiveness by inhibiting phosphorylation of EGFR, HER2, Akt, ERK, and p38, and down-regulating MMP-9 when compared to erlotinib. Afatinib abolished radiation-induced lung metastases in vivo. Furthermore, LLC HER2 knock-down cells treated with radiation had growth inhibition. CONCLUSION: Dual inhibition of radiation-activated EGFR and HER2 signaling by afatinib suppressed the proliferation and invasion of irradiated LLC cells. Increased radiosensitivity and decreased metastatic dissemination were observed by pharmacological or genetic HER2 inhibition in vivo. These findings indicate that HER2 plays a pivotal role in enhancing radioresistance and reducing metastatic potential of LLC cells.

Poly-ADP ribosylation in DNA damage response and cancer therapy.

Poly(ADP-ribosyl)ation (aka PARylation) is a unique protein post-translational modification (PTM) first described over 50 years ago. PARylation regulates a number of biological processes including chromatin remodeling, the DNA damage response (DDR), transcription, apoptosis, and mitosis. The subsequent discovery of poly(ADP-ribose) polymerase-1 (PARP-1) catalyzing DNA-dependent PARylation spearheaded the field of DDR. The expanding knowledge about the poly ADP-ribose (PAR) recognition domains prompted the discovery of novel DDR factors and revealed crosstalk with other protein PTMs including phosphorylation, ubiquitination, methylation and acetylation. In this review, we highlight the current knowledge on PAR-regulated DDR, PAR recognition domain, and PARP inhibition in cancer therapy.

Impact of androgen-deprivation therapy on the outcome of dose-escalation prostate cancer radiotherapy without elective pelvic irradiation.

The benefit of androgen-deprivation therapy (ADT) in combination with dose-escalated radiotherapy (DERT) for localized prostate cancer has not been determined in randomized studies. In this study, the benefit of ADT was assessed in patients uniformly treated with dose-escalated intensity-modulated radiation therapy (IMRT) to the prostate and seminal vesicles but not pelvis. In all, 419 patients with localized prostate adenocarcinoma underwent definitive IMRT (cumulative dose 78 Gy), with 32.6%, 33.1%, 32.1%, and 2.1% having T1 through T4 disease, respectively, and 51.2% having high-risk disease. ADT was given to 76.1% of patients. With a median follow-up of 60 months, 5-year biochemical failure-free, disease-free, and overall survival rates were 87%, 86%, and 87%, respectively. T stage was an independent predictor of all three rates. Five-year pelvic nodal recurrence rate was 2.9%. ADT improved biochemical failure-free and disease-free survival but not overall survival. ADT showed benefit in high-risk disease but not intermediate-risk disease. Late gastrointestinal and genitourinary toxicities ≥ grade 2 occurred in 11.0% and 6.7%, respectively. In conclusion, DERT with 78 Gy yields good disease control and low rate of pelvic nodal recurrence. ADT improves disease-free survival in patients with high-risk but not intermediate-risk disease.

Differences in toxicity and outcome associated with circadian variations between patients undergoing daytime and evening radiotherapy for prostate adenocarcinoma.

This retrospective study tested the hypothesis that disease control and treatment-related toxicity in patients undergoing high-dose radiotherapy (HDRT) for prostate cancer varies in a circadian manner. Patients with localized prostate adenocarcinoma receiving HDRT (median 78 Gy) to the prostate and involved seminal vesicle(s) without elective pelvic irradiation were divided into a daytime treatment (before 5 PM) group (n = 267) and evening treatment (after 5 PM) group (n = 142). Biochemical failure (Phoenix definition), acute and late gastrointestinal (GI) and genitourinary toxicities (Common Terminology Criteria for Adverse Events version 4), biochemical failure-free survival (BFFS) and freedom from late toxicity were assessed. Analyses were performed by binary logistic regression and Cox proportional hazard regression. The median follow-up was 68 months, and 75% of patients were ≥70 years old. Evening HDRT was significantly associated with worse freedom from ≥grade 2 late GI complications (hazard ratio = 2.96; p < 0.001). The detrimental effect of evening HDRT was significant in patients older than 70 years old (p < 0.001) but not in younger patients (p = 0.63). In a subgroup of propensity score-matched cohort with T2b-T3 disease (n = 154), the 5-year BFFS was worse in the evening group than the daytime group (72% vs. 85%, hazard ratio = 1.95, p = 0.05). Our study indicates that evening HDRT may lead to more GI complications, especially in older patients, and worse BFFS in patients with T2b-T3 disease.

Dual-timing PSA as a biomarker for patients with salvage intensity modulated radiation therapy for biochemical failure after radical prostatectomy.

We investigated the outcomes and the associated clinical-pathological factors in patients with prostate cancer (PCa) undergoing salvage intensity modulated radiation therapy (IMRT) for post-radical-prostatectomy (RP) biochemical failure. We report clinical outcomes of post-RP salvage IMRT, and describe chronic toxicity in these patients.Fifty patients with PCa underwent post-RP salvage IMRT. The median dose of IMRT was 70 Gy to the prostatic and seminal vesicle bed. Clinical-pathological and toxicity information were collected. The prostate cancer-specific survival (PCSS), disease-free survival (DFS), and biochemical-failure-free survival (BFFS) were calculated. Prognostic factors were analyzed for their association with disease control.The median follow-up time was 74 months. The 5-year PCSS, DFS, and BFFS after salvage IMRT were 95%, 88%, and 60%, respectively. Two patients (4%) experienced late gastrointestinal toxicity ≥ grade 3, and 5 patients (10%) had late genitourinary toxicity ≥ grade 3. On multivariate analysis, post-RP prostate-specific antigen (PSA) nadir ≤0.1 ng/ml (P=0.018) and PSA ≤0.5 ng/ml at salvage IMRT (P=0.016) were independent factors predicting better BFFS. Patients with both post-RP PSA nadir ≤0.1 ng/ml and PSA ≤0.5 ng/ml at salvage IMRT had a 5-year BFFS of 83% as compared with 43% in other patients (P=0.001).In conclusion, with hormonal therapy in most PCa patients, the addition of salvage IMRT for post-RP biochemical failure can achieve a good outcome with low toxicity. Patients with a post-RP PSA nadir ≤0.1 ng/ml and PSA ≤0.5 ng/ml at salvage IMRT could benefit the most from salvage IMRT.

Crystal engineering toward intersecting channels in a interpenetrated diamondoid network based on a net-to-net H-bonding interaction.

A thermally stable, four-fold interpenetrating diamondoid coordination network, Cd(imidazole-4-acrylate)2, with open intersecting channels within the interwoven nets, is strategically designed and synthesized on the basis of a spring-like net-to-net hydrogen-bonding interaction.

Adaptive radiation therapy for postprostatectomy patients using real-time electromagnetic target motion tracking during external beam radiation therapy.

PURPOSE: Using real-time electromagnetic (EM) transponder tracking data recorded by the Calypso 4D Localization System, we report inter- and intrafractional target motion of the prostate bed, describe a strategy to evaluate treatment adequacy in postprostatectomy patients receiving intensity modulated radiation therapy (IMRT), and propose an adaptive workflow. METHODS AND MATERIALS: Tracking data recorded by Calypso EM transponders was analyzed for postprostatectomy patients that underwent step-and-shoot IMRT. Rigid target motion parameters during beam delivery were calculated from recorded transponder positions in 16 patients with rigid transponder geometry. The delivered doses to the clinical target volume (CTV) were estimated from the planned dose matrix and the target motion for the first 3, 5, 10, and all fractions. Treatment adequacy was determined by comparing the delivered minimum dose (Dmin) with the planned Dmin to the CTV. Treatments were considered adequate if the delivered CTV Dmin is at least 95% of the planned CTV Dmin. RESULTS: Translational target motion was minimal for all 16 patients (mean: 0.02 cm; range: -0.12 cm to 0.07 cm). Rotational motion was patient-specific, and maximum pitch, yaw, and roll were 12.2, 4.1, and 10.5°, respectively. We observed inadequate treatments in 5 patients. In these treatments, we observed greater target rotations along with large distances between the CTV centroid and transponder centroid. The treatment adequacy from the initial 10 fractions successfully predicted the overall adequacy in 4 of 5 inadequate treatments and 10 of 11 adequate treatments. CONCLUSION: Target rotational motion could cause underdosage to partial volume of the postprostatectomy targets. Our adaptive treatment strategy is applicable to post-prostatectomy patients receiving IMRT to evaluate and improve radiation therapy delivery.

CRSBP-1/LYVE-1 ligands stimulate contraction of the CRSBP-1-associated ER network in lymphatic endothelial cells.

CRSBP-l/LYVE-1 ligands (PDGF-BB, VEGF-A(165) and hyaluronic acid) have been shown to induce opening of lymphatic intercellular junctions in vitro and in vivo by stimulating contraction of lymphatic endothelial cells (LECs). The mechanism by which CRSBP-1 ligands stimulate contraction of LECs is not understood. Here we demonstrate that CRSBP-1 is localized to the plasma membrane as well as intracellular fibrillar structures in LECs, including primary human dermal LECs and SVEC4-10 cells. CRSBP-1-associated fibrillar structures are identical to the ER network as evidenced by the co-localization of CRSBP-1 and BiP in these cells. CRSBP-1 ligands stimulate contraction of the ER network in a CRSBP-1-dependent and paclitaxel (a microtubule-stabilizing agent)-sensitive manner. These results suggest that ligand-stimulated ER contraction is associated with ligand-stimulated contraction in LECs.

Racial disparities in the use of voice preservation therapy for locally advanced laryngeal cancer.

OBJECTIVE: To identify potential racial disparities in the use of larynx preservation. DESIGN: Retrospective database review. SETTING: Academic medical center. PATIENTS: The Surveillance Epidemiology and End Results (SEER) database was used to identify white, black, Hispanic, and Asian patients with stage III and IV laryngeal cancers that were diagnosed during 1991 through 2008. Patients with T4 disease or distant metastasis were intentionally excluded. MAIN OUTCOME MEASURE: Univariate and multivariate logistic regression analysis, with odds ratios and 95% confidence intervals, was used to investigate the relationship between race/ethnicity and the use of larynx preservation with radiation therapy as initial therapy. RESULTS: Among the 5385 cases of laryngeal cancers that met the selection criteria, the racial distribution was white (72.7%), black (16.8%), Hispanic (7.4%), and Asian (3.1%). On univariate analysis, blacks (odds ratio [OR], 0.72; 95% CI, 0.59-0.88) were significantly less likely to undergo larynx preservation. This racial disparity persisted on multivariate analysis for blacks (OR, 0.78; 95% CI, 0.63-0.96) and was still observed among patients treated more recently between 2001 and 2008 (OR, 0.74; 95% CI, 0.56-0.96). CONCLUSIONS: Pronounced racial disparities exist in the use of larynx preservation therapy for locally advanced laryngeal cancer. While acknowledging the potential biases of socioeconomic factors, further research to better elucidate the underlying reasons for these findings may be warranted.

Inhibitors of clathrin-dependent endocytosis enhance TGFbeta signaling and responses.

Clathrin-dependent endocytosis is believed to be involved in TGFbeta-stimulated cellular responses, but the subcellular locus at which TGFbeta induces signaling remains unclear. Here, we demonstrate that inhibitors of clathrin-dependent endocytosis, which are known to arrest the progression of endocytosis at coated-pit stages, inhibit internalization of cell-surface-bound TGFbeta and promote colocalization and accumulation of TbetaR-I and SARA at the plasma membrane. These inhibitors enhance TGFbeta-induced signaling and cellular responses (Smad2 phosphorylation/nuclear localization and expression of PAI-1). Dynasore, a newly identified inhibitor of dynamin GTPase activity, is one of the most potent inhibitors among those tested and, furthermore, is a potent enhancer of TGFbeta. Dynasore ameliorates atherosclerosis in the aortic endothelium of hypercholesterolemic ApoE-null mice by counteracting the suppressed TGFbeta responsiveness caused by the hypercholesterolemia, presumably acting through its effect on TGFbeta endocytosis and signaling in vascular cells.

Rabbit erythrocytes possess adenylyl cyclase type II that is activated by the heterotrimeric G proteins Gs and Gi.

Adenosine triphosphate (ATP) release from rabbit erythrocytes occurs in response to deformation or reduced oxygen tension. A signal transduction pathway that relates these stimuli to ATP release has been proposed. This pathway includes the heterotrimeric G proteins, Gs and Gi, adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator. Importantly, adenylyl cyclase types II, IV and VII have been reported to be activated by both Gs and Gi. Here, we demonstrate that rabbit erythrocytes possess an adenylyl cyclase subtype that is activated both by the alpha subunit and the betagamma subunit of Gs and Gi, respectively. Washed rabbit erythrocytes released ATP when exposed to the beta adrenergic receptor-mediated activator of Gs, isoproterenol (ISO, 10 microM, n = 8, p < 0.05) as well as in response to incubation with a direct activator of Gi, mastoparan 7 (MAS7, 10 microM, n = 12, p < 0.05). In contrast, an inactive mastoparan derivative, mastoparan 17 (MAS 17, 10 microM, n = 6) did not stimulate ATP release. Importantly, incubation of washed rabbit erythrocytes with either isoprotenerol (ISO) (10 microM, n = 7) or MAS7 (10 microM, n = 11) resulted in increases in cyclic adenosine monophosphate (cAMP) (p < 0.01).Western analysis was used to determine if an adenylyl cyclase capable of being activated by both Gs and Gi was a component of rabbit erythrocyte membranes. We identified adenylyl cyclase type II with two antibodies generated against different epitopes of the protein. These results provide support for the hypothesis that, in rabbit erythrocytes, activation of either Gs or Gi results in the stimulation of adenylyl cyclase resulting in increases in cAMP leading, ultimately, to the release of ATP.