Eag1 Expression Interferes with Hypoxia Homeostasis and Induces Angiogenesis in Tumors

Abstract

Ether-á-go-go-1 (Eag1) is a CNS-localized voltage-gated potassium channel that is found ectopically expressed in a majority of extracranial solid tumors. While circumstantial evidence linking Eag1 to tumor biology has been well established, the mechanisms by which the channel contributes to tumor progression remain elusive. In this study, we have used in vivo and in vitro techniques to identify a candidate mechanism. A mutation that eliminates ion permeation fails to completely abolish xenograft tumor formation by transfected cells, indicating that Eag1 contributes to tumor progression independently of its primary function as an ion channel. Our data suggest that Eag1 interferes with the cellular mechanism for maintaining oxygen homeostasis, increasing HIF-1 activity, and thereby VEGF secretion and tumor vascularization. Ether-á-go-go-1 (Eag1) is a CNS-localized voltage-gated potassium channel that is found ectopically expressed in a majority of extracranial solid tumors. While circumstantial evidence linking Eag1 to tumor biology has been well established, the mechanisms by which the channel contributes to tumor progression remain elusive. In this study, we have used in vivo and in vitro techniques to identify a candidate mechanism. A mutation that eliminates ion permeation fails to completely abolish xenograft tumor formation by transfected cells, indicating that Eag1 contributes to tumor progression independently of its primary function as an ion channel. Our data suggest that Eag1 interferes with the cellular mechanism for maintaining oxygen homeostasis, increasing HIF-1 activity, and thereby VEGF secretion and tumor vascularization. Ether-à-go-go 1 (Eag1, 2The abbreviations used are: Eagether-à-go-goPBSphosphate-buffered salineVEGFvascular endothelial growth factorELISAenzyme-linked immunosorbent assayHIFhypoxia-inducible factor KCNH1, Kv10.1 (1Gutman G.A. Chandy K.G. Grissmer S. Lazdunski M. McKinnon D. Pardo L.A. Robertson G.A. Rudy B. Sanguinetti M.C. Stühmer W. Wang X. Pharmacol. Rev. 2005; 57: 473-508Crossref PubMed Scopus (697) Google Scholar)) is the founding member of the eag voltage-dependent potassium channel family, which also includes Erg and Elk channels (2Bauer C.K. Schwarz J.R. J. Membr. Biol. 2001; 182: 1-15Crossref PubMed Scopus (105) Google Scholar, 3Warmke J.W. Ganetzki B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar). The human Eag1 is normally expressed only within the nervous system (4Occhiodoro T. Bernheim L. Liu J.H. Bijlenga P. Sinnreich M. Bader C.R. Fischer-Lougheed J. FEBS Lett. 1998; 434: 177-182Crossref PubMed Scopus (119) Google Scholar, 5Pardo L.A. del Camino D. Sánchez A. Alves F. Brüggemann A. Beckh S. Stühmer W. EMBO J. 1999; 18: 5540-5547Crossref PubMed Scopus (364) Google Scholar), yet ectopic Eag1 expression has been increasingly linked to oncogenesis and tumor progression. Overexpressing Eag1 induces a phenotype with characteristic features of tumorigenic cells (5Pardo L.A. del Camino D. Sánchez A. Alves F. Brüggemann A. Beckh S. Stühmer W. EMBO J. 1999; 18: 5540-5547Crossref PubMed Scopus (364) Google Scholar). Additionally, Eag1 mRNA and its product have been detected in numerous cancer cell lines (6Meyer R. Heinemann S.H. J. Physiol. 1998; 508: 49-56Crossref PubMed Scopus (105) Google Scholar, 7Ouadid-Ahidouch H. Le Bourhis X. Roudbaraki M. Toillon R.A. Delcourt P. Prevarskaya N. Recept. Channels. 2001; 7: 345-356PubMed Google Scholar, 8Meyer R. Schonherr R. Gavrilova-Ruch O. Wohlrab W. Heinemann S.H. J. Membr. Biol. 1999; 171: 107-115Crossref PubMed Scopus (93) Google Scholar) and primary tumors (9Mello de Queiroz F. Suarez-Kurtz G. Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 42Crossref PubMed Scopus (87) Google Scholar, 10Hemmerlein B. Weseloh R.M. Queiroz F.M. d. Knötgen H. Sánchez A. Rubio M.E. Martin S. Schliephacke T. Jenke M. Heinz-Joachim-Radzun Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 41Crossref PubMed Scopus (208) Google Scholar, 11Farias L.M. B. Bermúdez Ocaña D. Díaz L. Larrea F. Avila-Chávez E. Cadena A. Hinojosa L.M. Lara G. Villanueva L.A. Vargas C. Hernández-Gallegos E. Camacho-Arroyo I. Dueñas-González A. Pérez-Cárdenas E. Pardo L.A. Morales A. Taja-Chayeb L. Escamilla J. Sánchez-Peña C. Camacho J. Cancer Res. 2004; 64: 6996-7001Crossref PubMed Scopus (122) Google Scholar). Finally, siRNA-mediated knockdown of Eag1 expression reduces proliferation (12Weber C. Mello de Queiroz F. Downie B. Sukow A. Stühmer W. Pardo L.A. J. Biol. Chem. 2006; 281: 13033-13037Abstract Full Text Full Text PDF Scopus (101) Google Scholar), and the inhibition of channel function reduces tumor progression both in vitro (7Ouadid-Ahidouch H. Le Bourhis X. Roudbaraki M. Toillon R.A. Delcourt P. Prevarskaya N. Recept. Channels. 2001; 7: 345-356PubMed Google Scholar, 13Gavrilova-Ruch O. Schönherr K. Gessner G. Schönherr R. Klapperstuck T. Wohlrab W. Heinemann S.H. J. Membr. Biol. 2002; 188: 137-149Crossref PubMed Scopus (109) Google Scholar) and in vivo (14Gomez-Varela D. Zwick-Wallasch E. Knotgen H. Sanchez A. Hettmann T. Ossipov D. Weseloh R. Contreras-Jurado C. Rothe M. Stuhmer W. Pardo L.A. Cancer Res. 2007; 67: 7343-7349Crossref PubMed Scopus (175) Google Scholar). Yet despite the clear involvement of Eag1 in proliferation and tumor progression, the mechanism of its transformative properties has remained a mystery. ether-à-go-go phosphate-buffered saline vascular endothelial growth factor enzyme-linked immunosorbent assay hypoxia-inducible factor Several other K+ channels have been implicated in tumor progression and cell proliferation (15Pardo L.A. Contreras-Jurado C. Zientkowska M. Alves F. Stuhmer W. J. Membr. Biol. 2005; 205: 115-124Crossref PubMed Scopus (169) Google Scholar, 16Pardo L.A. Physiology. 2004; 19: 285-292Crossref PubMed Scopus (259) Google Scholar, 17Conti M. J. Exp. Ther. Oncol. 2004; 4: 161-166PubMed Google Scholar). Different theories have attempted to explain the possible role of potassium channels in proliferation, all of which rely on the effects of increased potassium permeability (e.g. Refs. 18Rouzaire-Dubois B. Malo M. Dubois J.M. Rouzaire-Dubois B. Benoit E. Dubois J.M. Ion Channels and Physiopathologies of Nerve Conduction and Cell Proliferation. Research Signpost, Trivandrum, India2002: 187-200Google Scholar, 19Dubois J.M. Rouzaire-Dubois B. Eur. Biophys. J. 2004; 33: 227-232Crossref PubMed Scopus (45) Google Scholar, 20Wonderlin W.F. Strobl J.S. J. Membr. Biol. 1996; 154: 91-107Crossref PubMed Scopus (353) Google Scholar). Despite these efforts, a clear relationship between potassium permeation and tumor progression has yet to be established. In particular, the reason why only some channels can influence the behavior of tumor cells remains elusive (15Pardo L.A. Contreras-Jurado C. Zientkowska M. Alves F. Stuhmer W. J. Membr. Biol. 2005; 205: 115-124Crossref PubMed Scopus (169) Google Scholar, 16Pardo L.A. Physiology. 2004; 19: 285-292Crossref PubMed Scopus (259) Google Scholar, 17Conti M. J. Exp. Ther. Oncol. 2004; 4: 161-166PubMed Google Scholar, 21Stuhmer W. Alves F. Hartung F. Zientkowska M. Pardo L.A. FEBS Lett. 2006; 580: 2850-2852Crossref PubMed Scopus (76) Google Scholar). In this study, we investigated a possible interaction between Eag1 and signaling routes known to be relevant for tumor progression. The hypoxic intratumoral environment promotes the activation of hypoxia-inducible factor 1 (HIF-1), which is a key transcription factor influencing a broad spectrum of functions. Among them, neo-vascularization through transcriptional activation of vascular endothelial growth factor (VEGF) has become a promising target for cancer therapy as neoangiogenesis has a major influence on tumor progression (e.g. Ref. 22Collins I. Workman P. Nat. Chem. Biol. 2006; 2: 689-700Crossref PubMed Scopus (331) Google Scholar). In the presence of normal oxygen conditions, the regulated subunit of HIF-1 (HIF-1α; Refs. 23Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 24Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar) is rapidly degraded by the proteasome on an order of minutes (25Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4179) Google Scholar) in an ubiquitin-dependent manner. The E3 ubiquitin ligase responsible for this process contains pVHL (von-Hippel-Lindau tumor suppressing factor), a well-established tumor suppressor (26Kaelin Jr., W.G. Nat. Rev. Cancer. 2002; 2: 673-682Crossref PubMed Scopus (698) Google Scholar). Previous reports show an influence of pVHL in the functional expression of Eag1 in neuroblastoma cells (27Murata H. Tajima N. Nagashima Y. Yao M. Baba M. Goto M. Kawamoto S. Yamamoto I. Okuda K. Kanno H. Cancer Res. 2002; 62: 7004-7011PubMed Google Scholar), which was interpreted as a sign for differentiation to a neuronal phenotype. We designed the present study to elucidate a possible cross-talk between Eag1 and the hypoxia homeostasis system. Cell Culture—All parental cell lines were obtained from DSMZ (Germany) and maintained according to the instructions of the provider. Transient transfections were performed using Fugene (Roche Applied Science). Stable transfectants were selected and maintained using Zeocin (Calya, 3 μg/ml in culture medium). 24 h after transfection, transiently transfected cells were transferred to hypoxic conditions for 4 h. Culturing under hypoxia was performed by N2 injection in an oxygen-controlling chamber (Labotec). Flow Cytometry—Cells were incubated for 20 h in the absence of selective pressure, washed twice with cold PBS, and fixed for 30 min with 4% p-formaldehyde at 4 °C. Fixed cells were scraped from the flask, washed three times, and permeabilized with 0.3% saponin for 15 min at room temperature. Cells were then incubated with 10% bovine serum albumin, 0.15% saponin for 15 min to block nonspecific binding, and incubated for 2 h at 4°C with HIF-1a antibody (AB463, BD Biosciences) diluted 1:100 in 0.1% saponin/2% bovine serum albumin in PBS (PBS-SB). Cells were washed three times with PBS-SB and then incubated 1.5 h with a Cy5-conjugated anti-mouse antibody diluted 1:5000 in PBS-SB. Cells were again washed three times and assessed for Cy5 fluorescence using a BD FACSAria flow cytometer (Becton Dickinson, Heidelberg, Germany) with 633-nm laser excitation. Forward and side scatter were used to detect and discard cell fragments and doublets. Data are presented as average fluorescence. VEGF ELISA—Mock-transfected, Clone A, Clone B, and Clone C HEK293 cells were counted and plated at the same density in 25 cm2 flasks. Cells were cultured overnight with medium containing the selective agent Zeocin (3 μg/ml), after which the media was changed and cultured for 20 h without selection pressure. Cell culture supernatant was removed and centrifuged to remove cell particulates and detached cells, then assayed for secreted VEGF (Quantikine VEGF ELISA assay kit, R&D Systems). Cells in the flask were stained with propidium iodide and counted using a flow cytometer. Luciferase Assay—Mock-transfected and Clone A cells were counted and plated at the same density in 6-well plates. Cells were then transfected using Lipofectamine 2000 in Optimem with Bartrons HRE vector for 4 h, after which the medium was changed to normal growth medium. Luminescence was assayed using a Luciferase Reporter Gene Assay from Roche Applied Science. Western Blot—For HIF1α detection, cells were lysed in 10 mm Tris-HCl pH 6.8, 1% SDS, 5 mm dithiothreitol, Protease Inhibitor Mixture (Roche Applied Science), and 8 m urea and to minimize HIF degradation, homogenized using an UltraTurrax device, centrifuged (14,000 × g 15 min), and the supernatant was used as total cell extract. Protein concentration was determined using BCA (Pierce). Protein extracts were separated by gradient SDS-PAGE (either 3–8 or 4–12%) and transferred to nitrocellulose membranes. Membranes were blocked with 0.1% casein (Roche Applied Science) and incubated with the corresponding antibody. Antibody against HIF1α (BD Transduction Laboratories) was used at 0.5–1 μg/ml overnight in a humidified chamber. After washing and incubation with appropriate peroxidase-labeled secondary antibody, blots were developed using Millipore Immobilon system. Signals were detected in a Bio-Rad Chem-Doc luminescence detection system. Mouse Models—MDA-MB-435S (8 × 106), CHO (2 × 106), or NIH3T3 cells (6 × 106) were implanted into the flank of 8-week-old female scid were for 3 tumor growth inhibition after the tumors a of 2 mm in an was as in and diluted in phosphate-buffered saline to a 5 was to the using the same and tumor a were 2 minimize the same the tumors. was determined vivo at the of the and tumor were and in a of and was performed in a in 10 mm for 15 were incubated overnight in a humidified chamber at 4 with VEGF from or and was detected using the and were with and and in medium. The of vascular was performed by a using on 25 from of Eag1 Channels in inhibition of Eag1 by the channel and has been to proliferation of cell in vitro (7Ouadid-Ahidouch H. Le Bourhis X. Roudbaraki M. Toillon R.A. Delcourt P. Prevarskaya N. Recept. Channels. 2001; 7: 345-356PubMed Google Scholar, 13Gavrilova-Ruch O. Schönherr K. Gessner G. Schönherr R. Klapperstuck T. Wohlrab W. Heinemann S.H. J. Membr. Biol. 2002; 188: 137-149Crossref PubMed Scopus (109) Google Scholar), we effects be in CHO cells Eag1 were implanted into and tumors was selected of as the and have on growth by of cells was in the with to growth inhibition after with the well-established at the of 5 The of can be at to Eag1 as growth inhibition after of in CHO cell tumors We the of on the human cancer xenograft cell of Eag1 that is blocked by and has also been implicated in tumor progression A. 2005; PubMed Google was again to tumor progression as as without that ion permeation through Eag1 reduces tumor progression in in with using a antibody (14Gomez-Varela D. Zwick-Wallasch E. Knotgen H. Sanchez A. Hettmann T. Ossipov D. Weseloh R. Contreras-Jurado C. Rothe M. Stuhmer W. Pardo L.A. Cancer Res. 2007; 67: 7343-7349Crossref PubMed Scopus (175) Google Scholar). Ion by NIH3T3 Cells in of that potassium permeation is for Eag1 to tumor potassium permeation is the only of Eag1 channels relevant to tumor progression, of ion permeation a mutation in the also abolish tumor progression in these NIH3T3 and CHO cells were used as xenograft or NIH3T3 cells tumors implanted into cells Previous that tumors from the of CHO cells into are and by CHO cells (5Pardo L.A. del Camino D. Sánchez A. Alves F. Brüggemann A. Beckh S. Stühmer W. EMBO J. 1999; 18: 5540-5547Crossref PubMed Scopus (364) Google Scholar). Ion permeation was by a in the of the to has been to ion R. Gessner G. K. Heinemann S.H. FEBS Lett. 2002; PubMed Scopus Google Scholar). that the was and the channel was into and and channels to by that can be detected as by of channels to with properties with Eag1 Stable NIH3T3 and CHO transfectants of the were implanted in the flank of female While have that NIH3T3 cells to tumors in this cells were found to in with Eag1 tumors ion permeation the properties of is an were obtained CHO cells were used in of and channels tumors or is to that as an channel the channel in the the can to NIH3T3 cells, CHO cells also tumors. a of the of tumors from and with were in tumors of from or cells, the of these was increased in tumors Eag1 with the well-established endothelial the presence of endothelial cells and the as indicating that Eag1 expression the formation of vascular in tumors. Eag1 expression in xenograft tumors indicating that increased potassium of by of or in 25 from at tumors of increased vascular in both Eag1 and Eag1 tumors In of these a possible for the tumorigenic of Eag1 channels be increased in these tumors. the that to some the transformative properties of Eag1 are of its function as an ion channel and be on an with the cellular oxygen homeostasis system. oxygen and to tumor cells and a selective growth for Eag1 VEGF in the influencing VEGF to be B. J. M. N. N. Nature. PubMed Scopus Google Scholar, B. W. A. Nature. 1994; PubMed Scopus Google Scholar). VEGF has in the of both to its influence on tumor and to its in cancer the that the in of tumors to an in secreted we determined VEGF in the culture supernatant of cells on the cell lines used for xenograft increased VEGF secretion in cells both Eag1 and and for the of VEGF are normally to the human and on human and HEK293 cells were selected for human and discard to VEGF secretion was in three of the HEK293 cell Eag1 by ELISA A, B, and and with three cell lines were found to VEGF the VEGF with the of tumors. Eag1 candidate for the of VEGF in cells and in in tumors is a functional of this expression was using flow for HEK293 A, B, and C with an antibody and a secondary antibody. with cells, the fluorescence of a with an in The by Eag1 expression was under of the fluorescence for cell and to cells is in independently of oxygen concentration and culture conditions increased fluorescence this of can have functional we the of a HIF-1 was by the in both and HEK293 cells transiently transfected with a vector under of the HRE of M. C. X. J. M. F. Bartrons R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). cells maintained in a in with cells after h conditions by for h in in both and transfected cells, maintaining a in cells Western of cell extracts from to or 1% show in hypoxia 4 a clear in HIF1α in in HIF was under is that the in HIF can be to an A in HIF1α was in transiently transfected cells hypoxia 4 was 24 h after that Eag1 expression induces an in HIF1α under which a on the of the HIF-1 system. we determined the of conditions in the presence of of in Eag1 and cells were found to increased at of be interpreted as the of of Eag1 with the these suggest a relationship between the functional in and the of VEGF secretion in cell Despite ectopic expression of Eag1 in cancer cell lines and primary tumors (5Pardo L.A. del Camino D. Sánchez A. Alves F. Brüggemann A. Beckh S. Stühmer W. EMBO J. 1999; 18: 5540-5547Crossref PubMed Scopus (364) Google Scholar, 7Ouadid-Ahidouch H. Le Bourhis X. Roudbaraki M. Toillon R.A. Delcourt P. Prevarskaya N. Recept. Channels. 2001; 7: 345-356PubMed Google Scholar, 8Meyer R. Schonherr R. Gavrilova-Ruch O. Wohlrab W. Heinemann S.H. J. Membr. Biol. 1999; 171: 107-115Crossref PubMed Scopus (93) Google Scholar, de Queiroz F. Suarez-Kurtz G. Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 42Crossref PubMed Scopus (87) Google Scholar, 10Hemmerlein B. Weseloh R.M. Queiroz F.M. d. Knötgen H. Sánchez A. Rubio M.E. Martin S. Schliephacke T. Jenke M. Heinz-Joachim-Radzun Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 41Crossref PubMed Scopus (208) Google Scholar, 11Farias L.M. B. Bermúdez Ocaña D. Díaz L. Larrea F. Avila-Chávez E. Cadena A. Hinojosa L.M. Lara G. Villanueva L.A. Vargas C. Hernández-Gallegos E. Camacho-Arroyo I. Dueñas-González A. Pérez-Cárdenas E. Pardo L.A. Morales A. Taja-Chayeb L. Escamilla J. Sánchez-Peña C. Camacho J. Cancer Res. 2004; 64: 6996-7001Crossref PubMed Scopus (122) Google Scholar, C. Mello de Queiroz F. Downie B. Sukow A. Stühmer W. Pardo L.A. J. Biol. Chem. 2006; 281: 13033-13037Abstract Full Text Full Text PDF Scopus (101) Google Scholar, 21Stuhmer W. Alves F. Hartung F. Zientkowska M. Pardo L.A. FEBS Lett. 2006; 580: 2850-2852Crossref PubMed Scopus (76) Google Scholar), the mechanism of Eag1 to oncogenesis has remained a broad spectrum of we present evidence indicating that the of Eag1 is of its primary function as an ion channel. We also show a functional of HIF-1 and VEGF in We a possible mechanism by which Eag1 tumor progression through increased under the characteristic hypoxia of tumor Eag1 inhibition has in tumor cell proliferation in a mutation in Eag1 that ion permeation to tumor in NIH3T3 cells in that ion permeation is for the tumor of While at with the of a functional antibody to tumor progression (14Gomez-Varela D. Zwick-Wallasch E. Knotgen H. Sanchez A. Hettmann T. Ossipov D. Weseloh R. Contreras-Jurado C. Rothe M. Stuhmer W. Pardo L.A. Cancer Res. 2007; 67: 7343-7349Crossref PubMed Scopus (175) Google Scholar), this be the normally a of the Eag1 tumorigenic a blocked channel be in a the channel be to properties of Eag1 other ion permeation influence cellular as has been for eag Wilson Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar) and other voltage-gated channels Nat. Rev. 2006; 7: PubMed Scopus Google Scholar). The of ectopic expression of Eag1 in primary tumors (9Mello de Queiroz F. Suarez-Kurtz G. Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 42Crossref PubMed Scopus (87) Google Scholar, 10Hemmerlein B. Weseloh R.M. Queiroz F.M. d. Knötgen H. Sánchez A. Rubio M.E. Martin S. Schliephacke T. Jenke M. Heinz-Joachim-Radzun Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 41Crossref PubMed Scopus (208) Google Scholar) that Eag1 expression a selective to tumor cells under conditions that are to the majority of a be The major of hypoxia is HIF-1 which is as a of cancer both from of the tumor and the role of HIF-1 target in cell proliferation, and J. F. Nature. 2006; PubMed Scopus Google Scholar). of both Eag1 and HIF in in a phenotype in Ref. Rev. 2006; PubMed Scopus Google Scholar). The in HIF-1 in cells a for the of The ectopic expression of Eag1 have as the increased secretion of growth and as VEGF have effects within the tumor has been that the channel also VEGF secretion in cell the mechanism by which this is remains A. A. R. S. G. M. B. P. N. S. E. A. J. Cancer. 2005; PubMed Scopus Google Scholar). (27Murata H. Tajima N. Nagashima Y. Yao M. Baba M. Goto M. Kawamoto S. Yamamoto I. Okuda K. Kanno H. Cancer Res. 2002; 62: 7004-7011PubMed Google Scholar) a between pVHL and functional Eag1 is to that this interaction between the factor in of the of and Eag1 the in under hypoxia in cells, this In we have in this study a of an ion channel to tumor While have been as relevant for the in (e.g. Refs. Y.M. J. Y. N. M.C. Cancer Res. 2005; PubMed Scopus Google Scholar, J. A. J.M. Cancer. 2005; 5: PubMed Scopus Google Scholar), the evidence presented in this role in as We D. A. D. and F. for R. and D. for the HRE F. Mello de Queiroz and C. for in some S. for with the and for with