hrvatski

Utjecaj enkefalina i inhibitora enkefalinaze na stanice koštane srži bolesnika s leukemijom

Selektivni inhibitor membranske metaloendopeptidaze (imunofenotipskog obilježja CD10/CALLA, neutralne endopeptidaze - NEP) tiorfan dodavao se staničnim suspenzijama leukocitnog koncentrata koštane srži prije zasijavanja u u kratkotrajne (klonalne) kulture. Raspon koncentracija tiorfana bio je od 10^-5 M do 10^-13 M. Pratio se učinak na proliferaciju progenitorskih stanica GM-CFU u uzorcima koštane srži zdravog davatelja (6 eksperimenata), 10 bolesnika s ne-Hodgkinovim limfomom i 10 bolesnika s akutnom leukemijom. Većina bolesnika (17 od 20) bila je u kompletnoj, 2 od 20 u djelomičnoj remisiji bolesti, a 1 u relapsu. Učinkovitost zasijavanja stanica koštane srži bolesnika (tj. broj kolonija i skupina GM-CFU koje porastu u kulturi) znatno je varirala, odražavajući individualne razlike s obzirom na osnovnu bolest i provedenu antineoplastičku kemoterapiju. Stoga su se podaci iskazali u obliku postotaka broja kolonija i skupina u kontrolnim uzorcima koštane srži (tj. u uzorcima koji nisu tretirani tiorfanom). Na osnovi statističke analize distribucije broja kolonija i skupina u kulturama koštane srži zdravog davatelja i u kontrolnim uzorcima koštane srži bolesnika, pouzdanima su se smatrale promjene klonalnog rasta za 15% i više. Tiorfan je u nanomolarnim i višim koncentracijama poticao klonalni rast progenitorskih stanica GM-CFU u kratkotrajnim kulturama koštane srži zdravog davatelja. Djelotvorna koncentracija tiorfana bila je 10^-7 M. U kratkotrajnim kulturama 6 od 10 uzoraka koštane srži bolesnika s ne-Hodgkinovim limfomom u remisiji (3 centrocitna-centroblastna limfoma, 1 limfoblastični T-limfom, 2 anaplastična velikostanična limfoma CD30^+) tiorfan je poticao proliferaciju progenitorskih stanica GM-CFU. U 3 od 10 uzoraka (1 centrocitni B-stanični, 1 velikostanični anaplastični non-T non-B, 1 velikostanični centroblastni histiocitni NHL) tiorfan je sputavao proliferaciju GM-CFU, a u 1 uzorku (koštana srž bolesnika s anaplastičnim velikostaničnim NHL u relapsu) pokazao je učinak u oba smjera. Nije se uočila ovisnost učinka o koncentraciji tiorfana. Učinak tiorfana zadovoljavao je postavljene kriterije pouzdanosti u 31 od ukupno 79 kultura (39%). U tim su kulturama poticajni učinci (26) prevladavali nad inhibicijskima (5). Poticajni su se učinci češće očitovali u uzorcima koštane srži bolesnika s visokomalignim NHL, a uzorci koštane srži bolesnika s NHL nižeg stupnja malignosti bili su pretežno refrakterni na učinak tiorfana. Vjerodostojnost opažanja potvrđena je c^2-testom. Poticajni učinci tiorfana na klonalnu proliferaciju GM-CFU očitovali su se i u kratkotrajnim kulturama koštane srži bolesnika s akutnom leukemijom u remisiji. Upotrijebljeno je 7 uzoraka koštane srži bolesnika s ALL, 2 uzorka bolesnika s AML i 1 uzorak bolesnika s RAEB-t. U 2 od 10 uzoraka koštane srži (AML-M2 i ALL-L2) tiorfan je poticao proliferaciju GM-CFU u širokom rasponu koncentracija; u 5 od 10 uzoraka (2 od bolesnika s ALL-L1 i po 1 od ALL-L2, AML-M1-M2 i 1 RAEB-t) dobiveni su pretežno poticajni učinci ali i nešto supresivnih, ovisno o koncentraciji tiorfana; a u 3 od 10 uzoraka koštane srži (sva tri od bolesnika s ALL-L2) tiorfan nije pokazao mjerljiv učinak na proliferaciju GM-CFU. Poticajni su učinci tiorfana prevladavali u uzorcima koštane srži bolesnika s T-ALL i AL mijeloidnog tipa, a uzorci koštane srži bolesnika sa common ALL bili su pretežno refrakterni na učinak tiorfana. Učinak tiorfana zadovoljavao je postavljene kriterije pouzdanosti u 32 od ukupno 83 kultura (39%). U tim su kulturama poticajni učinci prevladavali nad inhibicijskima (24 prema 8). Vjerodostojnost opažanja potvrđena je c^2-testom. Tiorfansko djelovanje na krvotvorne stanice može se pripisati izravnim i neizravnim mehanizmima. Vezanje tiorfana na membransku metaloendopeptidazu CD10/NEP i internalizacija ili odbacivanje stvorenog kompleksa mogli bi pobuditi staničnu proliferaciju. Isti bi signal mogao potaknuti izlučivanje poticajnih citokina iz akcesornih stanica. A blokiranje enzimske aktivnosti membranske endopeptidaze (CD10/NEP) moglo bi smanjiti razgradnju citokina (IL-1, GM-CSF, G-CSF?) i drugih (neuropeptidnih?) poticatelja stanične proliferacije koji su nazočni u kultivacijskoj smjesi ili nastaju biosintezom u kultiviranim stanicama. U dugotrajnoj kulturi koštane srži osobe s akutnom limfoidnom leukemijom (ALL-L3) u remisiji nazočnost metioninskog enkefalina povećala je ukupan broj stanica u supernatantu i koncentraciju klonogenih GM-CFU u drugom tjednu kultiviranja. Promjene, premda očigledne, nisu dostigle razinu statističke signifikantnosti zbog premalenog broja uzoraka. U jednoj od kultura stromalni je sloj stanica bio vrlo oskudan, a u supernatantu je bilo mnogo više neatherentnih stanica nego u drugim kulturama. Međutim, klonogena moć tih stanica bila bitno manja od klonogene moći stanica u drugim kulturama. Aberantan stanični rast u toj kulturi pripisao se porastu leukemijskih stanica koje se na temelju morfoloških kriterija nisu mogle otkriti u punktatu koštane srži (minimalna rezidualna bolest). Opisani podaci govore u prilog pretpostavci da su neuropeptidi (metioninski enkefalin) i membranska endopeptidaza koja ih cijepa (CD10) uključeni u procese nadzora nad proliferacijom i diferencijacijom stanica krvotvornog tkiva. Poticajni učinci tiorfana u kratkotrajnim kulturama koštane srži normalnog davatelja i bolesnika s ne-Hodgkinovim limfomom ili akutnom leukemijom u remisiji upućuju na to da bi tiorfan i drugi inhibitori membranske metaloendopeptidaze mogli djelovati i na krvotvorno tkivo. Ti se spojevi pretklinički i klinički istražuju i pokušavaju upotrijebiti za suzbijanje boli i u terapiji astme, a već se upotrebljavaju za liječenje dijareje i hipertenzije. Opisane učinke inhibitora membranske neutralne metaloendopeptidaze na hematopoezu trebalo bi podrobnije istražiti i uzeti u obzir pri primjeni u bolesnika s neoplastičkim bolestima. 00981106 Summary Cell suspensions of the human bone marrow buffy coat were incubated with thiorphan, a selective inhibitor of the membrane metalloendopeptidase (the cell surface marker CD10/CALLA, neutral endopeptidase-NEP) before seeding into short-term clonal cultures. The bone marrow specimens were collected from a normal donor and used for 6 repeated experiments, from 10 patients with non-Hodgkin"s lymphoma and 10 patients with acute leukemia. Seventeen of the 20 patients were in complete and 2 in partial remission, and 1 was in the relapse.The concentration range of the agent was 10^-5 to 10^-13 M. Its effect on the granulocyte-macrophage colony count (GM-CFU) was determined. The seeding efficiency of the bone marrow specimens collected from the patients varied, reflecting the disease status, antineoplastic chemotherapy delivered, and the duration and quality of the remission. In order to make the results comparable, the colony counts in the thiorphan-treated cultures were expressed as the percentages of the counts in control cultures without the agent. Based on the statistical analysis of the data scatter, alterations of the clonal growth for 15 percent or more were considered to be meaningful. Thiorphan in nanomolar and higher concentrations stimulated the GM-CFU growth in clonal cultures of normal bone marrow. Significant increase was recorded in thiorphan concentration 10^-7 M. In 6 of 10 bone marrow samples of the patients with non-Hodgkin"s lymphoma in remission (3 centrocytic-centroblastic lymphomas, 1 lymphoblastic T-lymphoma, 2 anaplastic large cell lymphomas CD30^+) thiorphan stimulated the GM-CFU progenitor cell proliferation. In 3 of 10 samples (1 centrocytic B-lymphoma, 1 anaplastic large cell non-T non-B lymphoma, 1 large cell centroblastic histiocytic NHL) thiorphan inhibited the GM-CFU proliferation, and in 1 sample (the bone marrow of a patient with anaplastic large cell NHL in relapse) the stimulatory as well as the inhibitory effects were noted. There was no clear dose-effect relationship. Thiorphan effects complied with the effectiveness criteria in 31 cultures out of 79 (39 percent). In those cultures the stimulatory effects outnumbered the inhibitory ones (26 versus 5). The stimulatory effects were encountered more frequently in the bone marrow samples of the patients with highly malignant NHL, whereas the samples of the patients with NHLs of the medium malignancy grade were refractory to the thiorphan effects. The significance of the observations was confirmed by the c^2-test. The stimulatory effects of thiorphan on GM-CFU proliferation were also noted in clonal cultures of the bone marrow of the patients with acute leukemia in remission. The material included 7 samples of the patients with ALL, 2 with AML and 1 with RAEB-t. In 2 of 10 bone marrow samples (AML-M2 and ALL-L2) thiorphan stimulated the GM-CFU proliferation over a wide concentration range; in 5 of 10 samples (2 from the patients with ALL-L1 and 3 from the patients with ALL-L2, AML-M1-M2 or RAEB-t) thiorphan stimulated and, depending on the concentration, occassionally inhibited the clonal growth; and in 3 of 10 samples (all three from the patients with ALL-L2) no alteration was recorded. The stimulatory effects occurred predominantly in bone marrow samples of the patients with T-ALL or with myeloid-type AL, whereas the bone marrow cultures of the patients with "common"-type ALL were refractory to thiorphan effects. The criteria for the effectiveness of the treatment were fulfilled in 32 cultures out of 83 (39 percent). In those cultures the stimulatory effects outnumbered the inhibitory ones (24 versus 8). The significance of the observations was confirmed by the c^2-test. Thiorphan effects on the hematopoietic cells may be attributed to the direct and the indirect mechanisms. Cell proliferation could be triggered by thiorphan binding to the membrane metalloendopeptidase CD10/NEP followed by internalization or shedding of the formed complex. And the blocking of the endopeptidase (CD10/NEP) activity might reduce the enzymatic degradation of the cytokines (IL-1, GM-CSF, G-CSF?) and other (neuropeptide?) growth factors that are present in the culture medium or formed by the cultured cells. Opioid peptide methionine enkephalin was added to a long-term culture of bone marrow cells obtained from a patient with acute lymphoid leukemia (ALL-L3) in remission. The total cell count and the concentration of clonogenic GM-CFU in the supernatants showed an increase in the second week of cultivation. The alterations, although evident, did not reach the level of statistical significance because of the small number of the cultures. In one of the cultures the stromal layer was very scarce and the supernatant contained a high number of nonadherent cells with low colonogenic ability. That aberrant growth pattern was attributed to the proliferation of leukemia cells present in the original bone marrow sample but escaping detection by morphologic criteria (minimal residual disease). These observations support the idea that neuropeptides (methionine enkephalin) and the membrane endopeptidase(s) cleaving them participate in processes controlling the proliferation and differentiation of hematopoietic cells. Stimulatory effects of thiorphan on hematopoietic cell proliferation in the cultures of bone marrow obtained from normal donor and from patients with non-Hodgkin"s lymphoma or acute leukemia in remission indicate that thiorphan and related inhibitors of membrane neutral endopeptidase(s) intended for the treatment of pain and asthma and already used for the treatment of diarrhea and hypertension, might affect hematopoiesis. Those side-effects should be investigated in more detail and taken into account in possible applications of these drugs in patients afflicted with neoplastic disease.

hematopoeza; stanična kultura; leukemija; membranska metaloendopeptidaza; CD10; CALLA; tiorfan

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