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1. Preservation of Axillary Lymph Nodes Compared with Complete Dissection in T1-2 Breast Cancer Patients Presenting One or Two Metastatic Sentinel Lymph Nodes: The SINODAR-ONE Multicenter Randomized Clinical Trial

Author

Tinterri, C., Gentile, D., Gatzemeier, W., Sagona, A., Barbieri, E., Testori, A., Errico, V., Bottini, A., Marrazzo, E., Dani, C., Dozin, B., Boni, L., Bruzzi, P., Fernandes, B., Franceschini, D., Spoto, R., Torrisi, R., Scorsetti, M., Santoro, A., Canavese, G., Custodero, O., Troilo, V. L., Taffurelli, M., Cucchi, M. C., Galluzzo, V., Cabula, C., Cabula, R., Lazzaretti, M. G., Caruso, F., Castiglione, G., Grossi, S., Tavoletta, M. S., Rossi, C., Curcio, A., Friedman, D., Fregatti, P., Magni, C., Tazzioli, G., Papi, S., Giovanazzi, R., Chifu, C., Bettini, R., Pezzella, M., Michieletto, S., Saibene, T., Roncella, M., Ghilli, M., Sibilio, A., Cariello, A., Coiro, S., Falco, G., Meli, E. Z., Fortunato, L., Ciuffreda, L., Murgo, R., Battaglia, C., Rubino, L., Biglia, N., Bounous, V., Rovera, F. A., Chiappa, C., Pollini, G., Mirandola, S., Meneghini, G., and Di Bartolo, F.

Subjects
Sentinel Lymph Node Biopsy, Breast Neoplasms, Oncology, Lymphatic Metastasis, Axilla, Humans, Lymph Node Excision, Surgery, Female, Lymph Nodes, Prospective Studies, Neoplasm Recurrence, Local, Sentinel Lymph Node, Mastectomy
Abstract

The SINODAR-ONE trial is a prospective noninferiority multicenter randomized study aimed at assessing the role of axillary lymph node dissection (ALND) in patients undergoing either breast-conserving surgery or mastectomy for T1-2 breast cancer (BC) and presenting one or two macrometastatic sentinel lymph nodes (SLNs). The endpoints were to evaluate whether SLN biopsy (SLNB) only was associated with worsening of the prognosis compared with ALND in terms of overall survival (OS) and relapse.Patients were randomly assigned (1:1 ratio) to either removal of ≥ 10 axillary level I/II non-SLNs followed by adjuvant therapy (standard arm) or no further axillary treatment (experimental arm).The trial started in April 2015 and ceased in April 2020, involving 889 patients. Median follow-up was 34.0 months. There were eight deaths (ALND, 4; SNLB only, 4), with 5-year cumulative mortality of 5.8% and 2.1% in the standard and experimental arm, respectively (p = 0.984). There were 26 recurrences (ALND 11; SNLB only, 15), with 5-year cumulative incidence of recurrence of 6.9% and 3.3% in the standard and experimental arm, respectively (p = 0.444). Only one axillary lymph node recurrence was observed in each arm. The 5-year OS rates were 98.9% and 98.8%, in the ALND and SNLB-only arm, respectively (p = 0.936).The 3-year survival and relapse rates of T1-2 BC patients with one or two macrometastatic SLNs treated with SLNB only, and adjuvant therapy, were not inferior to those of patients treated with ALND. These results do not support the use of routine ALND.

Published
2022

5. 20-year risks of breast-cancer recurrence after stopping endocrine therapy at 5 years

Author

Pan, H, Gray, R, Braybrooke, J, Davies, C, Taylor, C, Mcgale, P, Peto, R, Pritchard, Ki, Bergh, J, Dowsett, M, Hayes, Df, Albain, K, Anderson, S, Arriagada, R, Barlow, W, Bartlett, J, Bergsten‐nordström, E, Bliss, J, Boccardo, F, Bradley, R, Brain, E, Cameron, D, Clarke, M, Coates, A, Coleman, R, Correa, C, Costantino, J, Cuzick, J, Davidson, N, Dodwell, D, Di Leo, A, Ewertz, M, Forbes, J, Gelber, R, Gnant, M, Goldhirsch, A, Goodwin, P, Hill, C, Ingle, J, Jagsi, R, Janni, W, Loibl, S, Mackinnon, E, Martin, M, Mukai, H, Norton, L, Ohashi, Y, Paik, S, Perez, E, Piccart, M, Pierce, L, Poortmans, P, Raina, V, Ravdin, P, Regan, M, Robertson, J, Rutgers, E, Slamon, D, Sparano, J, Swain, S, Tutt, A, Viale, G, Von Minckwitz, G, Wang, X, Whelan, T, Wilcken, N, Winer, E, Wolmark, N, Wood, W, Zambetti, M, Alberro, Ja, Ballester, B, Deulofeu, P, Fábregas, R, Fraile, M, Gubern, Jm, Janer, J, Moral, A, De Pablo Jl, Peñalva, G, Puig, P, Ramos, M, Rojo, R, Santesteban, P, Serra, C, Solà, M, Solarnau, L, Solsona, J, Veloso, E, Vidal, S, Abe, O, Abe, R, Enomoto, K, Kikuchi, K, Koyama, H, Masuda, H, Nomura, Y, Sakai, K, Sugimachi, K, Toi, M, Tominaga, T, Uchino, J, Yoshida, M, Haybittle, Jl, Leonard, Cf, Calais, G, Garaud, P, Collett, V, Delmestri, A, Sayer, J, Harvey, Vj, Holdaway, Im, Kay, Rg, Mason, Bh, Forbes, Jf, Balic, M, Bartsch, R, Fesl, C, Fitzal, F, Fohler, H, Greil, R, Jakesz, R, Marth, C, Mlineritsch, B, Pfeiler, G, Singer, Cf, Steger, Gg, Stöger, H, Canney, P, Yosef, Hma, Focan, C, Peek, U, Oates, Gd, Powell, J, Durand, M, Mauriac, L, Dolci, S, Larsimont, D, Nogaret, Jm, Philippson, C, Piccart, Mj, Masood, Mb, Parker, D, Price, Jj, Lindsay, Ma, Mackey, J, Hupperets, Psgj, Bates, T, Blamey, Rw, Chetty, U, Ellis, Io, Mallon, E, Morgan, Dal, Patnick, J, Pinder, S, Lohrisch, C, Nichol, A, Bramwell, Vh, Chen, Be, Gelmon, K, Goss, Pe, Levine, Mn, Parulekar, W, Pater, Jl, Shepherd, Le, Tu, D, Berry, D, Broadwater, G, Cirrincione, C, Muss, H, Weiss, Rb, Abu‐zahra, Ht, Portnoj, Sm, Bowden, S, Brookes, C, Dunn, J, Fernando, I, Lee, M, Poole, C, Rea, D, Spooner, D, Barrett‐lee, Pj, Mansel, Re, Monypenny, Ij, Gordon, Nh, Davis, Hl, Sestak, I, Lehingue, Y, Romestaing, P, Dubois, Jb, Delozier, T, Griffon, B, Mace Lesec’h, J, De La Lande, B, Mouret‐fourme, E, Mustacchi, G, Petruzelka, L, Pribylova, O, Owen, Jr, Harbeck, N, Jänicke, F, Meisner, C, Schmitt, M, Thomssen, C, Meier, P, Shan, Y, Shao, Yf, Zhao, Db, Chen, Zm, Howell, A, Swindell, R, Boddington, C, Burrett, Ja, Cutter, D, Duane, F, Evans, V, Gettins, L, Godwin, J, James, S, Kerr, A, Liu, H, Mannu, G, Mchugh, T, Morris, P, Read, S, Wang, Y, Wang, Z, Albano, J, De Oliveira Cf, Gervásio, H, Gordilho, J, Ejlertsen, B, Jensen, Mb, Johansen, H, Mouridsen, H, Palshof, T, Gelman, Rs, Harris, Jr, Henderson, C, Shapiro, Cl, Christiansen, P, Mouridsen, Ht, Fehm, T, Trampisch, Hj, Dalesio, O, De Vries Ege, Rodenhuis, S, Van Tinteren, H, Comis, Rl, Davidson, Ne, Robert, N, Sledge, G, Solin, Lj, Sparano, Ja, Tormey, Dc, Dixon, Jm, Forrest, P, Jack, W, Kunkler, I, Rossbach, J, Klijn, Jgm, Treurniet‐donker, Ad, Van Putten Wlj, Rotmensz, N, Veronesi, U, Bartelink, H, Bijker, N, Bogaerts, J, Cardoso, F, Cufer, T, Julien, Jp, Van De Velde Cjh, Cunningham, Mp, Brufsky, Am, Coleman, Re, Llombart, Ha, Huovinen, R, Joensuu, H, Costa, A, Bonadonna, G, Gianni, L, Valagussa, P, Goldstein, Lj, Bonneterre, J, Fargeot, P, Fumoleau, P, Kerbrat, P, Luporsi, E, Namer, M, Carrasco, E, Segui, Ma, Eiermann, W, Hilfrich, J, Jonat, W, Kaufmann, M, Kreienberg, R, Schumacher, M, Bastert, G, Rauschecker, H, Sauer, R, Sauerbrei, W, Schauer, A, Blohmer, Ju, Costa, Sd, Eidtmann, H, Gerber, B, Jackisch, C, De Schryver, A, Vakaet, L, Belfiglio, M, Nicolucci, A, Pellegrini, F, Pirozzoli, Mc, Sacco, M, Valentini, M, Mcardle, Cs, Smith, Dc, Stallard, S, Dent, Dm, Gudgeon, Ca, Hacking, A, Murray, E, Panieri, E, Werner, Id, Galligioni, E, Leone, B, Vallejo, Ct, Zwenger, A, Lopez, M, Erazo, A, Medina, Jy, Horiguchi, J, Takei, H, Fentiman, Is, Hayward, Jl, Rubens, Rd, Skilton, D, Scheurlen, H, Sohn, Hc, Untch, M, Dafni, U, Markopoulos, C, Fountzilas, G, Mavroudis, D, Klefstrom, P, Blomqvist, C, Saarto, T, Gallen, M, Tinterri, C, Margreiter, R, De Lafontan, B, Mihura, J, Roché, H, Asselain, B, Salmon, Rj, Vilcoq, Jr, André, F, Delaloge, S, Koscielny, S, Michiels, S, Rubino, C, A'Hern, R, Ellis, P, Kilburn, L, Yarnold, Jr, Benraadt, J, Kooi, M, Van De Velde Ao, Van Dongen Ja, Vermorken, Jb, Castiglione, M, Colleoni, M, Collins, J, Gelber, Rd, Lindtner, J, Price, Kn, Regan, Mm, Rudenstam, Cm, Senn, Hj, Thuerlimann, B, Bliss, Jm, Chilvers, Ced, Coombes, Rc, Hall, E, Marty, M, Buyse, M, Possinger, K, Schmid, P, Wallwiener, D, Bighin, C, Bruzzi, P, Del Mastro, L, Dozin, B, Pastorino, S, Pronzato, P, Sertoli, Mr, Foster, L, George, Wd, Stewart, Hj, Stroner, P, Borovik, R, Hayat, H, Inbar, Mj, Peretz, T, Robinson, E, Camerini, T, Formelli, F, Martelli, G, Di Mauro Mg, Perrone, F, Amadori, D, Martoni, A, Pannuti, F, Camisa, R, Musolino, A, Passalacqua, R, Iwata, H, Shien, T, Ikeda, T, Inokuchi, K, Sawa, K, Sonoo, H, Sadoon, M, Tulusan, Ah, Kohno, N, Miyashita, M, Takao, S, Ahn, Jh, Jung, Kh, Korzeniowski, S, Skolyszewski, J, Ogawa, M, Yamashita, J, Bastiaannet, E, Liefers, Gj, Christiaens, R, Neven, P, Paridaens, R, Van Den Bogaert, W, Braun, S, Martin, P, Romain, S, Janauer, M, Seifert, M, Sevelda, P, Zielinski, Cc, Hakes, T, Hudis, Ca, Wittes, R, Giokas, G, Kondylis, D, Lissaios, B, De La Huerta, R, Sainz, Mg, Ro, J, Camphausen, K, Danforth, D, Lichter, A, Lippman, M, Smart, D, Steinberg, S, D’Amico, C, Lioce, M, Paradiso, A, Ohno, S, Bass, G, Brown, A, Bryant, J, Dignam, J, Fisher, B, Geyer, C, Mamounas, Ep, Redmond, C, Wickerham, L, Aihara, T, Hozumi, Y, Baum, M, Jackson, Im, Palmer, Mk, Ingle, Jn, Suman, Vj, Bengtsson, No, Emdin, S, Jonsson, H, Venturini, M, Lythgoe, Jp, Kissin, M, Erikstein, B, Hannisdal, E, Jacobsen, Ab, Reinertsen, Kv, Varhaug, Je, Gundersen, S, Hauer‐jensen, M, Høst, H, Nissen‐meyer, R, Mitchell, Ak, Robertson, Jfr, Ueo, H, Di Palma, M, Mathé, G, Misset, Jl, Levine, M, Morimoto, K, Takatsuka, Y, Crossley, E, Harris, A, Talbot, D, Taylor, M, Cocconi, G, Di Blasio, B, Ivanov, V, Paltuev, R, Semiglazov, V, Brockschmidt, J, Cooper, Mr, Falkson, Ci, Hadji, P, A’Hern, R, Makris, A, Parton, M, Pennert, K, Powles, Tj, Smith, Ie, Gazet, Jc, Browne, L, Graham, P, Corcoran, N, Clack, G, Van Poznak, C, Deshpande, N, Di Martino, L, Douglas, P, Lindtner, A, Notter, G, Bryant, Ajs, Ewing, Gh, Firth, La, Krushen‐kosloski, Jl, Anderson, H, Killander, F, Malmström, P, Rydén, L, Arnesson, Lg, Carstensen, J, Dufmats, M, Fohlin, H, Nordenskjöld, B, Söderberg, M, Carpenter, Jt, Murray, N, Royle, Gt, Simmonds, Pd, Crowley, J, Gralow, J, Hortobagyi, G, Livingston, R, Martino, S, Osborne, Ck, Ravdin, Pm, Bondesson, T, Celebioglu, F, Dahlberg, K, Fornander, T, Fredriksson, I, Frisell, J, Göransson, E, Iiristo, M, Johansson, U, Lenner, E, Löfgren, L, Nikolaidis, P, Perbeck, L, Rotstein, S, Sandelin, K, Skoog, L, Svane, G, Af Trampe, E, Wadström, C, Maibach, R, Thürlimann, B, Holli, K, Rouhento, K, Safra, T, Brenner, H, Hercbergs, A, Yoshimoto, M, Paterson, Ahg, Fyles, A, Meakin, Jw, Panzarella, T, Bahi, J, Lemonnier, J, Martin, Al, Reid, M, Spittle, M, Bishop, H, Bundred, Nj, Forsyth, S, Pinder, Se, Deutsch, Gp, Kwong, Dlw, Pai, Vr, Senanayake, F, Rubagotti, A, Hackshaw, A, Houghton, J, Ledermann, J, Monson, K, Tobias, Js, Carlomagno, C, De Laurentiis, M, De Placido, S, Williams, L, Bell, R, Hinsley, S, Marshall, Hc, Pierce, Lj, Solomayer, E, Horsman, Jm, Lester, J, Winter, Mc, Buzdar, Au, Hsu, L, Love, Rr, Ahlgren, J, Garmo, H, Holmberg, L, Liljegren, G, Lindman, H, Wärnberg, F, Asmar, L, Jones, Se, Aft, R, Gluz, O, Liedtke, C, Nitz, U, Litton, A, Wallgren, A, Karlsson, P, Linderholm, Bk, Chlebowski, Rt, Caffier, H., Guided Treatment in Optimal Selected Cancer Patients (GUTS), Other departments, CCA - Cancer Treatment and Quality of Life, Radiotherapy, Pan, Hongchao, Gray, Richard, Braybrooke, Jeremy, Davies, Christina, Taylor, Carolyn, Mcgale, Paul, Peto, Richard, Pritchard, Kathleen I, Bergh, Jona, Dowsett, Mitch, Hayes, Daniel F, De Laurentiis, Michelino, MUMC+: MA Medische Oncologie (9), RS: GROW - R3 - Innovative Cancer Diagnostics & Therapy, and Interne Geneeskunde

Subjects
0301 basic medicine, Oncology, medicine.medical_treatment, Kaplan-Meier Estimate, law.invention, 0302 clinical medicine, Randomized controlled trial, law, Recurrence, Receptors, Neoplasm Metastasis, AMERICAN SOCIETY, Adjuvant, CLINICAL-PRACTICE GUIDELINE, Absolute risk reduction, Estrogen Antagonists, General Medicine, Estrogen Antagonist, CHEMOTHERAPY, Middle Aged, Prognosis, Neoplasm Metastasi, Local, POSTMENOPAUSAL WOMEN, Receptors, Estrogen, Chemotherapy, Adjuvant, 030220 oncology & carcinogenesis, Meta-analysis, Lymphatic Metastasis, Female, Human, Estrogen Antagonists/therapeutic use, Adult, Risk, medicine.medical_specialty, Prognosi, medicine.drug_class, DISCONTINUATION, Breast Neoplasms, Article, Drug Administration Schedule, LATE DISTANT RECURRENCE, 03 medical and health sciences, Breast cancer, Breast Neoplasms/drug therapy, Internal medicine, SCORE, medicine, Humans, SURGICAL ADJUVANT BREAST, Aged, Proportional Hazards Models, Chemotherapy, business.industry, Proportional hazards model, Lymphatic Metastasi, TAMOXIFEN THERAPY, ta3122, medicine.disease, Estrogen, RANDOMIZED-TRIALS, Discontinuation, Surgery, Neoplasm Recurrence, 030104 developmental biology, Proportional Hazards Model, Neoplasm Grading, Neoplasm Recurrence, Local, business
Abstract

Background The administration of endocrine therapy for 5 years substantially reduces recurrence rates during and after treatment in women with early-stage, estrogen-receptor (ER)–positive breast cancer. Extending such therapy beyond 5 years offers further protection but has additional side effects. Obtaining data on the absolute risk of subsequent distant recurrence if therapy stops at 5 years could help determine whether to extend treatment. Methods In this meta-analysis of the results of 88 trials involving 62,923 women with ER-positive breast cancer who were disease-free after 5 years of scheduled endocrine therapy, we used Kaplan–Meier and Cox regression analyses, stratified according to trial and treatment, to assess the associations of tumor diameter and nodal status (TN), tumor grade, and other factors with patients’ outcomes during the period from 5 to 20 years. Results Breast-cancer recurrences occurred at a steady rate throughout the study period from 5 to 20 years. The risk of distant recurrence was strongly correlated with the original TN status. Among the patients with stage T1 disease, the risk of distant recurrence was 13% with no nodal involvement (T1N0), 20% with one to three nodes involved (T1N1–3), and 34% with four to nine nodes involved (T1N4–9); among those with stage T2 disease, the risks were 19% with T2N0, 26% with T2N1–3, and 41% with T2N4–9. The risk of death from breast cancer was similarly dependent on TN status, but the risk of contralateral breast cancer was not. Given the TN status, the factors of tumor grade (available in 43,590 patients) and Ki-67 status (available in 7692 patients), which are strongly correlated with each other, were of only moderate independent predictive value for distant recurrence, but the status regarding the progesterone receptor (in 54,115 patients) and human epidermal growth factor receptor type 2 (HER2) (in 15,418 patients in trials with no use of trastuzumab) was not predictive. During the study period from 5 to 20 years, the absolute risk of distant recurrence among patients with T1N0 breast cancer was 10% for low-grade disease, 13% for moderate-grade disease, and 17% for high-grade disease; the corresponding risks of any recurrence or a contralateral breast cancer were 17%, 22%, and 26%, respectively. Conclusions After 5 years of adjuvant endocrine therapy, breast-cancer recurrences continued to occur steadily throughout the study period from 5 to 20 years. The risk of distant recurrence was strongly correlated with the original TN status, with risks ranging from 10 to 41%, depending on TN status and tumor grade. (Funded by Cancer Research UK and others.)

Published
2017

6. Outcomes of hormone-responsive (HR+) HER2 negative (HER2-) metastatic breast cancer (MBC) patients (P) according to their starting first-line (1st) treatment (T): chemotherapy (CT) or hormonal therapy (HT)

Author

Bighin, C., primary, Poggio, F., additional, Dozin, B., additional, Ceppi, M., additional, Bruzzi, P., additional, D'Alonzo, A., additional, Giraudi, S., additional, Levaggi, A., additional, Lambertini, M., additional, Miglietta, L., additional, Vaglica, M., additional, Fontana, V., additional, Iacono, G., additional, Pronzato, P., additional, and Del Mastro, L., additional

Published
2015
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8. CTLA-4 expressed by human dendritic cells modulates thei cytokine secretion and induction of T cell proliferation

Author

Laurent, S., primary, Carrega, P., additional, Saverino, D., additional, Piccioli, P., additional, Camoriamo, M., additional, Morabito, A., additional, Dozin, B., additional, Fontana, V., additional, Simone, R., additional, Mortara, L., additional, Mingari, M.C., additional, Ferlazzo, G., additional, and Pistillo, M.P., additional

Published
2011
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17. Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro

Author

Castagnola, P, Dozin, B, Moro, G, and Cancedda, R

Abstract

This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary, types II and IX collagen mRNAs sharply increased within the first week of suspension culture, peaked in the second week, and thereafter began to decrease. This decrease was particularly significant for type IX collagen mRNA. The level of type X collagen mRNA progressively increased during the course of the culture, reached its maximal value after 3-4 wk, and decreased only at a later stage of cell differentiation. As determined by in vitro run-off transcription assays, all these changes in collagen mRNA levels could be attributed to parallel modifications in the relative rate of transcription of the corresponding collagen genes. We suggest that chicken chondrocyte differentiation proceeds through at least two different steps: (a) first, transition from a stage characterized by a high level of type I collagen mRNA to a stage characterized by predominance of types II and IX collagen mRNAs; (b) later, transition to a stage characterized by the highest level of type X collagen mRNA.

Published
1988
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18. Regulation of specific rat liver messenger ribonucleic acids by triiodothyronine.

Author

Magnuson, M A, Dozin, B, and Nikodem, V M

Abstract

A plasmid cDNA library was constructed using poly(A+) RNA isolated from the livers of rats treated with 3,5,3'-triiodothyronine (T3) and fed a high carbohydrate diet. This library was screened by differential colony hybridization with [32P]cDNA probes made from hypothyroid and hyperthyroid rat liver poly(A+) RNA to obtain clones representing T3-inducible mRNAs. Using plasmid cDNAs to 4 different T3-inducible mRNAs, we have studied by hybridization assay the responses of these mRNAs to different thyroidal steady states and to a high carbohydrate diet. The fold of induction (hypothyroid to hyperthyroid) varied from about 4.0 (mRNA 5-8D) to 13.2 (mRNA 4-12B). The linearity of response with regard to nuclear receptor occupancy was estimated by assessing the relative mRNA levels in a euthyroid state. Three of the mRNAs demonstrated nonlinear responses with the largest portion of the induction occurring in the euthyroid to hyperthyroid transition. An induction by the high carbohydrate diet was clearly seen for only one mRNA (5-8D) suggesting that these two pathways of induction are independent. In a study of the response kinetics of each mRNA to a nuclear receptor saturating dose of T3 in hypothyroid animals, an increase was seen within 4 h (the earliest time point examined) for one of the mRNAs. The other 3 mRNAs did not increase significantly until 8 h after the T3 dose. Northern analysis showed a single mRNA corresponding to each of these 4 clones with sizes ranging from about 1375 to 7600 bases. Two mRNAs (5-9E and 4-12B) were shown by hybrid-selected translation to code for proteins of molecular mass of about 27 and 46 kDa, respectively. The availability of several different cDNA probes to T3 responsive liver mRNAs should facilitate future studies on the mechanism of action of this hormone.

Published
1985
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19. Tissue-specific control of rat malic enzyme activity and messenger RNA levels by a high carbohydrate diet.

Author

Dozin, B, Rall, J E, and Nikodem, V M

Abstract

In euthyroid rats fed a high carbohydrate fat-free diet for 10 days, the mass of cellular malic enzyme mRNA in liver is increased 7- to 8-fold above the basal level. Malic enzyme activity is stimulated to the same extent. This effect does not result from an increase either in the transcriptional activity of the malic enzyme gene, as determined by nuclear run-off transcription assay, or in the content of intranuclear malic enzyme RNA sequences. Mathematical modeling shows that this increase in cytoplasmic mRNA is compatible with retarded degradation of cytoplasmic mRNA. Regulation of malic enzyme by carbohydrates is liver-specific, since no response is observed in the following nonhepatic tissues: brain, heart, spleen, kidney, testis, and lung. Furthermore, the amplitude of the response in liver depends on the thyroid state of the animals, being lower (by a factor of approximately 4) in hypothyroidism and higher (12- to 15-fold) when normal animals are injected simultaneously with a daily dose of 15 micrograms of triiodothyronine per 100 g of body weight for 10 days. Since thyroid hormones regulate liver malic enzyme synthesis predominantly at the nuclear level and carbohydrates at the cytoplasmic level, the additive effect of triiodothyronine and a high carbohydrate diet on the activity of malic enzyme is readily explicable.

Published
1986
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20. Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone.

Author

Song, M K, Dozin, B, Grieco, D, Rall, J E, and Nikodem, V M

Abstract

One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3′-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.

Published
1988
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26. Expression of the cartilage matrix protein gene at different chondrocyte developmental stages

Author

Muratoglu S, Bachrati C, Malpeli M, Piroska Szabó, Neri M, Dozin B, Deák F, Cancedda R, and Kiss I

Subjects
Extracellular Matrix Proteins, Cell Differentiation, Chick Embryo, Blotting, Northern, Immunohistochemistry, Kinetics, Cartilage, Gene Expression Regulation, Animals, Matrilin Proteins, RNA, Messenger, Cells, Cultured, Glycoproteins, Plasmids
Abstract

Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.

28. Chondrocyte differentiation in vitro from clones of prechondrogenic cells

Author

Rodolfo Quarto, Beatrice Dozin, Ranieri Cancedda, Giuliano Campanile, Carlo Tacchetti, Fiorella Descalzi, Cancedda, R., Quarto, R., Dozin, B., Descalzi, F., Tacchetti, Carlo, and Campanile, G.

Subjects
medicine.anatomical_structure, History and Philosophy of Science, Chemistry, General Neuroscience, medicine, General Biochemistry, Genetics and Molecular Biology, Chondrocyte, In vitro, Cell biology
Published
1990

29. In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells

Author

Beatrice Dozin, Ranieri Cancedda, Claudio Malfatto, Giuliano Campanile, Carlo Tacchetti, Rodolfo Quarto, Quarto, R, Dozin, B, Tacchetti, Carlo, Campanile, G, Malfatto, C, and Cancedda, R.

Subjects
Cellular differentiation, Type II collagen, Fluorescent Antibody Technique, Chick Embryo, Biology, Chondrocyte, medicine, Animals, RNA, Messenger, Cells, Cultured, Cell Aggregation, Cartilage, Embryo, Cell Differentiation, Cell Biology, Articles, Molecular biology, Cell aggregation, Clone Cells, Culture Media, medicine.anatomical_structure, Cell culture, Immunology, Collagen, Type I collagen
Abstract

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.

Published
1990

30. Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone

Author

Beatrice Dozin, Domenico Grieco, M.-K. H. Song, J. E. Rall, Vera M. Nikodem, Song, M. K. H., Dozin, B., Grieco, Domenico, Rall, J. E., and Nikodem, V. M.

Subjects
Regulation of gene expression, Messenger RNA, Malic enzyme, Intron, food and beverages, RNA, Cell Biology, Biology, Biochemistry, Molecular biology, Transcription (biology), Gene expression, Precursor mRNA, Molecular Biology
Abstract

One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3'-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.

Published
1988
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31. CTLA-4 expressed by chemoresistant,as well as untreated, myeloid leukemia cells can be targeted with ligands to induce apoptosis

Author

Maria Pia Pistillo, Beatrice Dozin, Marino Clavio, Giulio Lelio Palmisano, Pier Luigi Tazzari, Andrea Bacigalupo, Stefania Laurent, Alberto M. Martelli, Ivana Pierri, Tomohiro Kato, Giuseppe Balbi, Mauro Megna, Teresa Lamparelli, Marco De Gobbi, Anna Morabito, Laurent S, Palmisano GL, Martelli AM, Kato T, Tazzari PL, Pierri I, Clavio M, Dozin B, Balbi G, Megna M, Morabito A, Lamparelli T, Bacigalupo A, Gobbi M, and Pistillo MP

Subjects
Adult, Male, Programmed cell death, medicine.medical_specialty, Transcription, Genetic, Apoptosis, chemical and pharmacologic phenomena, Biology, Ligands, Myeloid Neoplasm, law.invention, Flow cytometry, Antigens, CD, Antigens, Neoplasm, law, hemic and lymphatic diseases, Internal medicine, Tumor Cells, Cultured, medicine, Humans, Cytotoxic T cell, CTLA-4 Antigen, Aged, Hematology, medicine.diagnostic_test, Reverse Transcriptase Polymerase Chain Reaction, hemic and immune systems, Middle Aged, Antigens, Differentiation, Drug Resistance, Neoplasm, Leukemia, Myeloid, CTLA-4, Caspases, Immunology, B7-1 Antigen, Recombinant DNA, Cancer research, Female, B7-2 Antigen
Abstract

We have previously reported that about 80% of acute myeloid leukaemia (AML) samples tested at diagnosis constitutively expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). The present study compared CTLA-4 expression and function of leukaemic cells from AML patients at diagnosis with those from AML patients resistant to conventional chemotherapy. We also explored the possibility of targeting CTLA-4 for apoptosis induction in chemoresistant AML cells. AML cells either from untreated patients (n = 15) or in chemoresistant phase (n = 10) were analysed for CTLA-4 protein and transcript expression by flow cytometry and reverse transcription-polymerase chain reaction respectively. CTLA-4 expression was similar in untreated and in chemoresistant samples and was not associated with patients' clinical features. In chemoresistant AML cells, CTLA-4 transduced an apoptotic signal on engagement with its recombinant ligands r-CD80 and r-CD86, which induced an average of 71% and 62% apoptotic cells, respectively, at highest concentration. Apoptosis was equally induced in untreated leukaemic cells accompanied by cleavage of procaspase-8 and -3. Thus, this study provides the first evidence that killing of leukaemic cells from AML patients may be obtained by the engagement of CTLA-4 with its ligands, opening the way to a novel potential therapeutic approach based on triggering the CTLA-4 molecule to circumvent chemoresistance in AML.

32. Assessment of interferon-γ in pleural fluid as a prognostic factor of survival in malignant pleural mesothelioma.

Author

Dozin B, Carbotti G, Roncella S, Ferro P, Dessanti P, Canessa PA, Ferrini S, and Fabbi M

Subjects
Adult, Aged, Cytokines analysis, Female, Humans, Male, Mesothelioma, Malignant immunology, Mesothelioma, Malignant mortality, Middle Aged, Pleural Effusion, Malignant immunology, Pleural Neoplasms immunology, Pleural Neoplasms mortality, Prognosis, Retrospective Studies, Interferon-gamma metabolism, Mesothelioma, Malignant pathology, Pleural Effusion, Malignant metabolism, Pleural Neoplasms pathology
Abstract

Backgound: Literature reports suggest that the host immune system may control Malignant Pleural Mesothelioma (MPM) growth, although its activity is limited by regulatory mechanisms. In this retrospective study, we analyzed the levels of pro-inflammatory (IL-1, IL-6, TNF), immune-regulatory (IL-10) and Th1/CTL-related cytokines (IL-12p70, IFN-γ) in the pleural exudate and their relationship with overall survival (OS) in MPM., Methods: Cytokines were quantified by multiplexed immunoassay. Concentrations were dichotomized with respect to the median value. Correlation between cytokine level and OS was assessed using univariate (Kaplan-Meier curves) and multivariate (Cox regression) analyses., Results: Regarding outcome, tumor histology, therapies undergone and IFN-γ were independent prognostic factors of OS in a 72 MPM training cohort. Notably, high concentrations of IFN-γ halved death probability (HR of high vs low IFN-γ concentration = 0.491, 95%CI 0.3-0.8, p = 0.007). Also in patients with epithelioid histology and those receiving at least one line of therapy, high IFN-γ level was an independent factor predictive of OS (HR of high vs low IFN-γ concentration were 0.497, p = 0.007 and 0.324, p = 0.006, respectively). However, these data were not confirmed in a 77 MPM validation cohort, possibly due to the low IFN-γ levels encountered in this population, and the heterogeneous distribution of disease stages between the training and the validation cohorts. None of the other cytokines showed any effect on survival., Conclusions: High level of IFN-γ in pleural effusion may be associated with better survival in MPM patients and potentially serve as a prognostic biomarker. Larger prospective studies are needed to ascertain this hypothesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Published
2021
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33. IL-27 Mediates PD-L1 Expression and Release by Human Mesothelioma Cells.

Author

Carbotti G, Dozin B, Martini S, Giordano C, Scordamaglia F, Croce M, Filaci G, Ferrini S, and Fabbi M

Abstract

Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Published
2021
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34. Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4 + tumor infiltrating lymphocytes and their in situ localization.

Author

Mastracci L, Fontana V, Queirolo P, Carosio R, Grillo F, Morabito A, Banelli B, Tanda E, Boutros A, Dozin B, Gualco M, Salvi S, Romani M, Spagnolo F, Poggi A, and Pistillo MP

Subjects
Adult, Aged, Aged, 80 and over, Antineoplastic Agents, Immunological therapeutic use, CD8-Positive T-Lymphocytes drug effects, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, CTLA-4 Antigen immunology, CTLA-4 Antigen metabolism, Female, Humans, Killer Cells, Natural drug effects, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Male, Melanoma immunology, Melanoma metabolism, Middle Aged, Retrospective Studies, Tumor Microenvironment immunology, CTLA-4 Antigen antagonists & inhibitors, Ipilimumab therapeutic use, Lymphocytes, Tumor-Infiltrating drug effects, Melanoma drug therapy, Tumor Microenvironment drug effects
Abstract

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4 + subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3 + cells. TILs and TIL subsets, such as CD3 + , CD45RO + , CTLA-4 + , CD4 + , CD8 + T cells, CD20 + B cells, and NKp46 + NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3 + , CD8 + T cells, and CTLA-4 + immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4 + TIL distribution in melanoma tissues taking into account localization, relationship with CD3 + /CD8 + TILs, and changes in response to IPI treatment. We identified that CTLA-4 + TILs may represent a marker of IPI response, alone or with CD3 + /CD8 + subsets, although this requires confirmation in larger studies.

Published
2020
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35. NEONOD 2: Rationale and design of a multicenter non-inferiority trial to assess the effect of axillary surgery omission on the outcome of breast cancer patients presenting only micrometastasis in the sentinel lymph node after neoadjuvant chemotherapy.

Author

Tinterri C, Canavese G, Bruzzi P, and Dozin B

Abstract

Sentinel lymph node biopsy alone, without complete axillary lymph node dissection, is the standard treatment of the axilla nodal chain in early-stage breast cancer patients presenting a negative sentinel lymph node. The updated results of the IBCSG 23-01 randomized trial recently provided evidence that this approach could be extended to early-stage breast cancer patients presenting only micrometastasis in the sentinel lymph node. On the other hand, patients with large operable or locally advanced breast cancer and clinically positive lymph nodes currently receive neoadjuvant chemotherapy and sentinel lymph node biopsy, which is then followed by complete axillary node dissection if the sentinel lymph node till contains tumor residue, regardless of the extent of nodal disease. Assuming that patients presenting only a micrometastatic sentinel lymph node after neoadjuvant chemotherapy are clinically equivalent to the IBCSG 23-01 early-breast cancer patients with only micrometastatic sentinel node, then complete axillary dissection would be unneeded also in these subset of patients in the neoadjuvant setting. The multicenter uncontrolled non-inferiority trial NEONOD 2 we here present was designed to assess this hypothesis, i.e. whether or not omission of complete axillary nodal clearance worsens prognosis in patients with sentinel node resulting only micrometastatic after neoadjuvant chemotherapy., Competing Interests: None., (© 2019 The Author(s).)

Published
2019
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36. Soluble CTLA-4 as a favorable predictive biomarker in metastatic melanoma patients treated with ipilimumab: an Italian melanoma intergroup study.

Author

Pistillo MP, Fontana V, Morabito A, Dozin B, Laurent S, Carosio R, Banelli B, Ferrero F, Spano L, Tanda E, Ferrucci PF, Martinoli C, Cocorocchio E, Guida M, Tommasi S, De Galitiis F, Pagani E, Antonini Cappellini GC, Marchetti P, Quaglino P, Fava P, Osella-Abate S, Ascierto PA, Capone M, Simeone E, Romani M, Spagnolo F, and Queirolo P

Subjects
Adult, Aged, Aged, 80 and over, Antineoplastic Agents, Immunological therapeutic use, Biomarkers, Tumor blood, CTLA-4 Antigen blood, Female, Humans, Italy, Kaplan-Meier Estimate, Male, Melanoma metabolism, Melanoma pathology, Middle Aged, Neoplasm Metastasis, Predictive Value of Tests, Solubility, Young Adult, Biomarkers, Tumor metabolism, CTLA-4 Antigen metabolism, Ipilimumab therapeutic use, Melanoma drug therapy
Abstract

CTLA-4 blockade by means of ipilimumab (IPI) potentiates the immune response and improves overall survival (OS) in a minority of metastatic melanoma (MM) patients. We investigated the role of soluble CTLA-4 (sCTLA-4) as a possible biomarker for identifying this subset of patients. sCTLA-4 levels were analyzed at baseline in sera from 113 IPI-treated MM patients by ELISA, and the median value (200 pg/ml) was used to create two equally sized subgroups. Associations of sCTLA-4 with best overall response (BOR) to IPI and immune-related adverse events (irAEs) were evaluated through logistic regression. Kaplan-Meier and Cox regression methods were used to analyze OS. A remarkable association between sCTLA-4 levels and BOR was found. Specifically, the proportion of patients with sCTLA-4 > 200 pg/ml in irSD or irPD (immune-related stable or progressive disease) was, respectively, 80% (OR = 0.23; 95%CL = 0.03-1.88) and 89% (OR = 0.11; 95%CL = 0.02-0.71) and was lower than that observed among patients in irCR/irPR (immune-related complete/partial response). sCTLA-4 levels increased during IPI treatment, since the proportion of patients showing sCTLA > 200 pg/ml after 3 cycles was 4 times higher (OR = 4.41, 95%CL = 1.02-19.1) than that after 1 cycle. Moreover, a significantly lower death rate was estimated for patients with sCTLA-4 > 200 pg/ml (HR = 0.61, 95%CL = 0.39-0.98). Higher baseline sCTLA-4 levels were also associated with the onset of any irAE (p value = 0.029), in particular irAEs of the digestive tract (p value = 0.041). In conclusion, our results suggest that high sCTLA-4 serum levels might predict favorable clinical outcome and higher risk of irAEs in IPI-treated MM patients.

Published
2019
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37. Corrigendum: Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

Author

Queirolo P, Dozin B, Morabito A, Banelli B, Piccioli P, Fava C, Leo C, Carosio R, Laurent S, Fontana V, Ferrucci PF, Martinoli C, Cocorocchio E, Battaglia A, Ascierto PA, Capone M, Simeone E, De Galitiis F, Pagani E, Antonini Cappellini GC, Marchetti P, Guida M, Tommasi S, Mandalà M, Merelli B, Quaglino P, Fava P, Guidoboni M, Romani M, Spagnolo F, and Pistillo MP

Abstract

[This corrects the article on p. 386 in vol. 8, PMID: 28446908.].

Published
2018
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38. Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Author

Muraglia A, Nguyen VT, Nardini M, Mogni M, Coviello D, Dozin B, Strada P, Baldelli I, Formica M, Cancedda R, and Mastrogiacomo M

Abstract

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Published
2017
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39. Hormonal therapy followed by chemotherapy or the reverse sequence as first-line treatment of hormone-responsive, human epidermal growth factor receptor-2 negative metastatic breast cancer patients: results of an observational study.

Author

Bighin C, Dozin B, Poggio F, Ceppi M, Bruzzi P, D'Alonzo A, Levaggi A, Giraudi S, Lambertini M, Miglietta L, Vaglica M, Fontana V, Iacono G, Pronzato P, and Del Mastro L

Subjects
Adult, Aged, Aged, 80 and over, Antineoplastic Agents, Hormonal administration & dosage, Biomarkers, Tumor, Breast Neoplasms metabolism, Breast Neoplasms mortality, Female, Follow-Up Studies, Humans, Kaplan-Meier Estimate, Middle Aged, Neoplasm Metastasis, Neoplasm Staging, Observational Studies as Topic, Odds Ratio, Receptor, ErbB-2 metabolism, Tomography, X-Ray Computed, Treatment Outcome, Antineoplastic Agents, Hormonal therapeutic use, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Breast Neoplasms diagnosis, Breast Neoplasms drug therapy
Abstract

Introduction Although hormonal-therapy is the preferred first-line treatment for hormone-responsive, HER2 negative metastatic breast cancer, no data from clinical trials support the choice between hormonal-therapy and chemotherapy.Methods Patients were divided into two groups according to the treatment: chemotherapy or hormonal-therapy. Outcomes in terms of clinical benefit and median overall survival (OS) were retrospectively evaluated in the two groups. To calculate the time spent in chemotherapy with respect to OS in the two groups, the proportion of patients in chemotherapy relative to those present in either group was computed at every day from the start of therapy.Results From 1999 to 2013, 119 patients received first-line hormonal-therapy (HT-first group) and 100 first-line chemotherapy (CT-first group). Patients in the CT-first group were younger and with poorer prognostic factors as compared to those in HT-first group. Clinical benefit (77 vs 81%) and median OS (50.7 vs 51.1 months) were similar in the two groups. Time spent in chemotherapy was significantly longer during the first 3 years in CT-first group (54-34%) as compared to the HT-first group (11-18%). This difference decreased after the third year and overall was 28% in the CT-first group and 18% in the HT-first group.Conclusions The sequence first-line chemotherapy followed by hormonal-therapy, as compared with the opposite sequence, is associated with a longer time of OS spent in chemotherapy. However, despite the poorer prognostic factors, patients in the CT-first group had a superimposable OS than those in the HT-first group.

Published
2017
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40. Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

Author

Queirolo P, Dozin B, Morabito A, Banelli B, Piccioli P, Fava C, Leo C, Carosio R, Laurent S, Fontana V, Ferrucci PF, Martinoli C, Cocorocchio E, Battaglia A, Ascierto PA, Capone M, Simeone E, De Galitiis F, Pagani E, Antonini Cappellini GC, Marchetti P, Guida M, Tommasi S, Mandalà M, Merelli B, Quaglino P, Fava P, Guidoboni M, Romani M, Spagnolo F, and Pistillo MP

Abstract

Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan-Meier method. Both -1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A "dose" (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, -1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying -1577G/A and CT60G/A, respectively. MM patients carrying -1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.

Published
2017
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42. Pulmonary middle lobectomy for non-small-cell lung cancer: effectiveness and prognostic implications.

Author

Mazza F, Ferrari E, Maineri P, Venturino M, Dozin B, and Ratto GB

Subjects
Adult, Aged, Aged, 80 and over, Carcinoma, Non-Small-Cell Lung diagnosis, Carcinoma, Non-Small-Cell Lung mortality, Female, Humans, Kaplan-Meier Estimate, Logistic Models, Lung Neoplasms diagnosis, Lung Neoplasms mortality, Lymph Node Excision mortality, Male, Middle Aged, Prognosis, Proportional Hazards Models, Retrospective Studies, Survival Analysis, Treatment Outcome, Young Adult, Carcinoma, Non-Small-Cell Lung surgery, Lung Neoplasms surgery, Pneumonectomy methods
Abstract

Objectives: The therapeutic value of pulmonary middle lobectomy (PML) has been questioned. PML is currently regarded as a standard form of lobectomy, even so it shares some surgical features with segmentectomies (SEG) more than with lobectomies. The present study's aim was to assess the therapeutic value of PML with respect to other lobectomies (LOBs) and SEGs., Methods: A total of 902 consecutive patients who underwent lobectomy or SEG with mediastinal lymph node dissection for Stage I-IIIa non-small-cell lung cancer were analysed. Patients with pT4 tumours and/or pathologically incomplete resection were excluded., Results: PML was performed in 50 patients, SEG in 44 and LOBs were performed in 808. The three study groups were homogeneous, except for gender, pT and grade: females, pT1 and G1 tumours were more frequent in the PML and SEG groups. The lymph node dissection yield was poorer in PML (P < 0.007) and SEG (P < 0.001) groups when compared with LOB group. Five-year overall survival (OS) was 45.3% for PML, 54.0% for SEG and 60.2% for LOB (P = 0.793). When limiting the analysis to G2-3 right-sided tumours, 5-year survival was lower in the PML group than in the LOB group: 41.3 vs 59.0% (P = 0.057). Similar results were found when analysing pT2-3 right-sided tumours: 27.3 vs 57.3% (P = 0.049). Multivariable analysis showed four independent prognostic factors: age (P = 0.001), pathological stage (P < 0.001), gender (P = 0.005) and the type of surgical resection (P = 0.029). PML (hazard ratio, HR = 1.63) and SEG (HR = 1.64) were detrimental in comparison with LOB. After adjusting for baseline differences between groups (propensity score), a trend towards a worse OS in PML group when compared with LOB group was observed (HR = 1.38, P = 0.150)., Conclusions: Both the lymphadenectomy yield and prognosis make PML more similar to SEG than lobectomy, especially for pT2-3 or G2-3 tumours., (© The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Published
2015
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43. Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

Author

Boero S, Morabito A, Banelli B, Cardinali B, Dozin B, Lunardi G, Piccioli P, Lastraioli S, Carosio R, Salvi S, Levaggi A, Poggio F, D'Alonzo A, Romani M, Del Mastro L, Poggi A, and Pistillo MP

Subjects
Adult, Aged, Aged, 80 and over, Antineoplastic Agents therapeutic use, Breast Neoplasms metabolism, Case-Control Studies, Cell Line, Tumor, Cohort Studies, Female, Flow Cytometry, Gene Frequency, Genotype, Humans, Immunohistochemistry, K562 Cells, Leukocytes, Mononuclear cytology, MCF-7 Cells, Middle Aged, Neoplasm Metastasis, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Antibody-Dependent Cell Cytotoxicity immunology, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Receptor, ErbB-2 metabolism, Receptors, IgG genetics, Trastuzumab therapeutic use
Abstract

Background: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab., Patients and Methods: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab., Results: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006)., Conclusions: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

Published
2015
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44. Incidence of hepatitis in patients with evidence of past or current hepatitis B or C during chemotherapy for early breast cancer.

Author

Levaggi A, De Maria A, Dozin B, Iacono G, Bighin C, Giraudi S, Lambertini M, D'Alonzo A, Poggio F, Pronzato P, and Del Mastro L

Subjects
Adult, Aged, Aged, 80 and over, Breast Neoplasms epidemiology, Case-Control Studies, Female, Hepatitis B blood, Hepatitis B immunology, Hepatitis B Core Antigens blood, Hepatitis B Surface Antigens blood, Hepatitis C, Chronic blood, Hepatitis C, Chronic immunology, Humans, Incidence, Italy epidemiology, Liver physiopathology, Middle Aged, Retrospective Studies, Breast Neoplasms drug therapy, Breast Neoplasms virology, Hepatitis B physiopathology, Hepatitis C, Chronic physiopathology
Abstract

Aim/background: Few data are available about the prevalence of hepatitis B and C infections in early breast cancer patients and its impact on systemic treatments. The objectives of this study were to determine the incidence of positive serology for hepatitis B and C in women with early breast cancer and to assess the clinical course and its impact on liver function during adjuvant treatments., Patients and Methods: we retrospectively reviewed hepatitis B and C serology [HBs antigen (HBsAg), HBc antibodies (HBcAb), HBs antibodies (HBsAb) and HC (HCV) antibodies] in 746 consecutive patients with early breast cancer treated at our Institution between 2009 and 2011., Results: Among 375 evaluable patients, we identified 312 controls (83.2%) and 63 patients (16.8%) with positive serology (cases): 15 patients (4%) with HCV, 8 (2.1%) with resolved HBV without anti-HBs (HBsAg-negative, HBsAgAb-negative, HBcAgAb-positive), 36 (9.6%) with resolved HBV with anti-HBs (HBsAg-negative, HBsAgAb-positive, HBcAgAb-positive) and 4 (1%) with chronic HBV (HBsAg-positive, HBsAgAb-negative, HBcAgAb-positive). During systemic treatments, hepatitis (defined as at least a three-fold increase in serum alanine aminotransferase level) occurred in nine (20.4%) out of 44 evaluable cases and in 14 (5.9%) out of 234 evaluable controls., Conclusion: Approximately 20% of patients with early breast cancer with positive serology for viral hepatitis may develop hepatitis during systemic treatment. Pre-treatment serum detection of viral hepatitis B and C antigens and antibodies may be useful in the adjuvant therapy decision-making process and for adequate monitoring of liver function during antineoplastic therapy., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Published
2014

45. CTLA-4 +49A>G polymorphism of recipients of HLA-matched sibling allogeneic stem cell transplantation is associated with survival and relapse incidence.

Author

Piccioli P, Balbi G, Serra M, Morabito A, Lamparelli T, Gobbi M, Laurent S, Dozin B, Bruzzi P, Ferraris AM, Bacigalupo A, Notaro R, and Pistillo MP

Subjects
Adolescent, Adult, Aged, CTLA-4 Antigen, Female, Hematopoietic Stem Cell Transplantation statistics & numerical data, Histocompatibility Testing, Humans, Male, Middle Aged, Myelodysplastic-Myeloproliferative Diseases epidemiology, Myelodysplastic-Myeloproliferative Diseases genetics, Recurrence, Retrospective Studies, Survival Analysis, Transplantation, Homologous, Young Adult, Antigens, CD genetics, Hematopoietic Stem Cell Transplantation adverse effects, Myelodysplastic-Myeloproliferative Diseases mortality, Myelodysplastic-Myeloproliferative Diseases therapy, Polymorphism, Single Nucleotide physiology, Siblings
Abstract

Conflicting observations have been reported about the role of CTLA-4 gene polymorphisms in the clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT). We have investigated three polymorphisms of the CTLA-4 gene (-318C>T, +49A>G, CT60G>A) in 133 donor/recipient pairs who underwent HLA-matched sibling donor HSCT for hematological malignancies. We found no association of the clinical outcome of the HSCT with either recipient or donor -318C>T and CT60G>A polymorphisms. At variance, we found a significant association of donor +49A>G G/G genotype with longer overall survival (OS; log-rank test, P = 0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P = 0.027), longer disease-free survival (P = 0.036) and reduced relapse rate (P = 0.042). However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT.

Published
2010
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46. Is there a subset of patients with preoperatively diagnosed N2 non-small cell lung cancer who might benefit from surgical resection?

Author

Ratto GB, Costa R, Maineri P, Alloisio A, Bruzzi P, and Dozin B

Subjects
Aged, Carcinoma, Non-Small-Cell Lung diagnosis, Carcinoma, Non-Small-Cell Lung pathology, Female, Humans, Lung Neoplasms diagnosis, Lung Neoplasms pathology, Lymph Node Excision, Male, Middle Aged, Pneumonectomy, Prognosis, Carcinoma, Non-Small-Cell Lung surgery, Lung Neoplasms surgery
Abstract

Objective: The role of surgery in the treatment of preoperatively diagnosed N2 non-small cell lung cancer remains controversial. This study sought significant prognostic factors to select candidates for surgery and assess prognosis., Methods: The study population included 277 patients who underwent primary resection (192) or induction chemotherapy followed by surgery (85) for preoperatively diagnosed, potentially resectable N2 non-small cell lung cancer. N2 descriptors were prospectively recorded. Kaplan-Meier curves were used to evaluate survival, and statistical significance of differences between curves was assessed by log-rank test. Cox regression was used for multivariate analyses., Results: Preoperative significant prognostic factors were number of mediastinal node levels involved (P < .001), symptom severity (P = .013), clinical T (P = .041), and induction chemotherapy (P = .001). Three groups with different prognoses were based on individual prognostic score. The group that did best had a median survival of 29.6 months. Postoperative predictors of survival were pathologic T (P = .003), tumor residue (P = .034), and number of mediastinal nodes involved (P < .001). Of 3 groups with different prognoses, the most favorable had a median survival as long as 42 months., Conclusion: This study provides a practical tool that uses significant prognostic factors to predict which patients with preoperatively diagnosed N2 non-small cell lung cancer have better prognoses. Because patients with the favorable prognostic factors showed good long-term survival and excellent local disease control, surgery should still play an important role in the multimodality treatment of these patients.

Published
2009
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47. Phylogeny and regulation of four lipocalin genes clustered in the chicken genome: evidence of a functional diversification after gene duplication.

Author

Pagano A, Giannoni P, Zambotti A, Sánchez D, Ganfornina MD, Gutiérrez G, Randazzo N, Cancedda R, and Dozin B

Subjects
Amino Acid Sequence, Animals, Base Sequence, Cell Size physiology, Cells, Cultured, Chondrocytes cytology, Chondrocytes drug effects, Chondrocytes metabolism, Evolution, Molecular, Exons, Gene Duplication, Gene Expression drug effects, Genes genetics, Genetic Variation genetics, Introns, Molecular Sequence Data, Promoter Regions, Genetic genetics, Sequence Homology, Amino Acid, Triiodothyronine pharmacology, Chickens genetics, Genome, Multigene Family genetics, Phylogeny, Proteins genetics
Abstract

A novel lipocalin gene is here reported that represents the fourth member of a cluster we have identified in the chicken genome. This cluster also includes Chondrogenesis-Associated Lipocalins beta and gamma (CAL beta, CAL gamma) and Extracellular Fatty Acid Binding Protein (Ex-FABP). The new gene codes for a 22-kDa secreted protein with three cysteine residues and a series of sequence features well conserved in the lipocalin family. All the genes in the cluster are structurally similar presenting comparable exon/intron boundary positions and exon sizes. A phylogenetic analysis indicates the monophyletic grouping of these genes, and their relationship with the lipocalins alpha-1-microglobulin (A1mg), complement factor 8 gamma chain (C8GC), prostaglandin D synthase (PGDS), and neutrophil-gelatinase-associated lipocalin (NGAL). The new cluster gene appears to be the ortholog of the mammalian C8GC and was thus named Ggal-C8GC. This orthology also suggests that this lipocalin was present in the ancestor common to reptiles and mammals. In addition to other expressing tissues, Ex-FABP, CAL beta and CAL gamma genes are highly transcribed in chondrocytes at late stages of chondrogenesis during endochondral bone formation and/or upon inflammatory stimulation. Here, we show that they are also transcriptionally induced when chondrocytes are subjected to various biological events as cell quiescence, cell shape transition, and hormonal stimulation. By contrast, Ggal-C8GC transcripts are only barely detectable in chondrocytes, but are more abundant in liver, kidney, brain, heart, skeletal muscle and particularly in skin. Moreover, no expression induction was observed neither during chondrocyte differentiation, nor upon any of the stimulations mentioned above. This indicates that the Ggal-C8GC gene was co-opted for a novel function after the duplication events that gave rise to the cluster. The peculiar coordinated regulation of Ex-FABP, CAL beta and CAL gamma, and the apparent divergent role of Ggal-C8GC suggest that these gene duplications may have been maintained during evolution by a sub-functionalization mechanism where some common function(s) are shared by several members of the cluster and some other specialized function(s) are unique to other members.

Published
2004
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48. A chondrogenesis-related lipocalin cluster includes a third new gene, CALgamma.

Author

Pagano A, Crooijmans R, Groenen M, Randazzo N, Zerega B, Cancedda R, and Dozin B

Subjects
Amino Acid Sequence, Animals, Brain embryology, Brain metabolism, Cell Differentiation genetics, Cell Division genetics, Cells, Cultured, Chick Embryo, Chondrocytes cytology, Chondrocytes metabolism, Collagen genetics, DNA chemistry, DNA genetics, Gene Expression, Gene Expression Regulation, Developmental, In Situ Hybridization, Liver embryology, Liver metabolism, Molecular Sequence Data, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Tibia embryology, Tibia metabolism, Time Factors, Carrier Proteins genetics, Chickens genetics, Multigene Family genetics
Abstract

We have previously reported the modulation, during chondrogenesis and/or inflammation, of two chicken genes laying in the same genomic locus and coding for two polypeptides of the lipocalin protein family, the extracellular fatty acid binding protein (ExFABP) and the chondrogenesis associated lipocalin beta (CALbeta). A third gene, located within the same cluster and coding for a new lipocalin, CALgamma, has been identified and is here characterized. Tissue distribution analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction in chicken embryos shows a ubiquitous expression with predominant levels of mRNA transcripts in the liver and the brain. In the developing tibia, a high expression of CALgamma mRNA was evidenced by in situ hybridization within the pre-hypertrophic and the hypertrophic zones of the bone-forming cartilage. In agreement, dedifferentiated chondrocytes in vitro express the transcripts to the highest level when they re-differentiate reaching hypertrophy. Such peculiar developmental pattern of expression that is analogous to those already described for Ex-FABP and CALbeta suggests that all three proteins may act synergistically in the process of endochondral bone formation. Moreover, like Ex-FABP and CALbeta, CALgamma is also highly induced in dedifferentiated chondrocytes upon stimulation with lypopolysaccharides, indicating that the whole cluster quite possibly is transcriptionally activated not only in physiological morphogenic differentiation but also in pathological acute phase response.

Published
2003
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49. Extracellular fatty acid binding protein (ex-FABP) is a stress protein expressed during chondrocyte and myoblast differentiation.

Author

Descalzi Cancedda F, Dozin B, Zerega B, Cermelli S, and Cancedda R

Subjects
2,2'-Dipyridyl pharmacology, Animals, Cells, Cultured, Chelating Agents pharmacology, Chick Embryo, Enzyme Inhibitors pharmacology, Fatty Acid-Binding Proteins, Hydrogen Peroxide pharmacology, Interleukin-6 physiology, Lipopolysaccharides pharmacology, Nitric Oxide Donors pharmacology, Oxidative Stress physiology, Penicillamine pharmacology, Carrier Proteins physiology, Cell Differentiation physiology, Chondrocytes cytology, Muscles cytology, Neoplasm Proteins
Abstract

Objective: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions., Design: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms., Results: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium., Conclusions: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

Published
2001

50. A divergent role of COOH-terminal domains in Nurr1 and Nur77 transactivation.

Author

Castillo SO, Xiao Q, Kostrouch Z, Dozin B, and Nikodem VM

Subjects
3T3 Cells, Alitretinoin, Alternative Splicing, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Chromosome Mapping, Cloning, Molecular, DNA metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Mice, Molecular Sequence Data, Nerve Tissue Proteins genetics, Nuclear Proteins metabolism, Nuclear Receptor Subfamily 4, Group A, Member 1, Nuclear Receptor Subfamily 4, Group A, Member 2, Receptors, Cytoplasmic and Nuclear, Receptors, Retinoic Acid metabolism, Receptors, Steroid chemistry, Receptors, Steroid genetics, Retinoid X Receptors, Structure-Activity Relationship, Transcription Factors chemistry, Transcription Factors genetics, Tretinoin metabolism, DNA-Binding Proteins metabolism, Nerve Tissue Proteins metabolism, Receptors, Steroid metabolism, Transcription Factors metabolism, Transcriptional Activation
Abstract

Orphan nuclear receptors such as Nurr1 and Nur77 have conserved amino acid sequences in the zinc finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. These receptors can bind DNA sequences (response elements) as monomers and can also heterodimerize with the retinoid X receptor to activate transcription. We report here the identification and initial characterization of a novel COOH-terminal truncated isoform of Nurr1, Nurr1a. Internal splicing of Nurr1 generates a frameshift such that a stop codon is prematurely encoded resulting in a naturally occurring COOH-terminal truncation. Embryonic and postnatal mouse brain showed both Nurr1 and Nurr1a mRNAs expressed during development. To characterize essential COOH-terminal elements that may be deleted from Nurr1a and determine function in putative ligand binding, we created COOH-terminal deletion mutants. Nurr1, Nur77, and 3'-truncated mutants bind in gel mobility shift assays to the monomeric Nur77 response element (B1A-RE). However, in transient transfection assays, a truncation of as little as 15 Nurr1 COOH-terminal amino acids diminished transcriptional activation of B1A-thymidine kinase-chloramphenicol acetyltransferase reporter. This result was not seen for a similar Nur77 deletion mutant, Nur77-586. Unlike full-length Nurr1 and Nur77, transactivation by Nur77-586 was not augmented in response to the presence of retinoid-like receptor and 9-cis-retinoic acid. Thus, the interaction of putative ligand binding and transactivation for Nurr1 and Nur77 may function differently.

Published
1998

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