Mechanisms of B-cell lymphoma development

Mehanizmi koji kontroliraju normalnu diferencijaciju i aktivaciju B-stanice često su u stanici limfoma kako bi u konačnici transformirana stanica imala neograničen rast i razvoj. B-stanice osobito su podložne malignim transformacijama zato što procesi koji dovode do velike raznolikosti imunoglobulina, koja je potrebna u normalnom imunološkom odgovoru, često vode do kromosomskih translokacija i onkogenih mutacija koje uključuju jake promotore Ig gena i mnoge tumor supresorske gene, kao i onkogene. Takvi događaji često vode do blokiranja diferencijacije, sprječavanja apoptoze i/ili poticanja proliferacije B-stanice koja konačno može biti prvi događaj u tranformaciji B-stanice u tumorsku stanicu. Dodatno, mnogi B-stanični limfomi trebaju ekspresiju BCR-a kako bi preživjeli, u nekim limfomima aktivacija stanice preko signalnog puta B-staničnog receptora te u nekima specifični faktori mikrookoliša tumora imaju važnu ulogu u tumorogenezi. Istraživanja na polju strukturne i funkcionalne genomike u zadnjih 10 godina značajno su doprinijela spoznajama o mehanizmima uključenim u patogenezu B-limfoma. Opisani su brojni signalni putovi koji su poremećeni u B-staničnim limfomima i koji vode do konsititutivne aktivacije brojnih drugih putova koji potiču preživljavanje tumorske stanice. Također, ovakav pristup istraživanja doveo je do klasifikacije B-limfoma prema stanicama od kojih su nastali budući da se zna kako su transformirane tumorske stanice u genetičkim i imunofenotipskim karakteristikama nalik nekom od tipova normalnih B-stanica. U godinama koje dolaze, nagli napredak genomike mogao bi dovesti do otkrića još mnogih drugih mehanizama koji su uključeni u transformaciju normalne B-stanice, što bi moglo pomoći u razvoju što uspješnijeg liječenja limfoma.Mechanisms that control normal B-cell differentiation and activation are frequently disrupted in lymphoma cells which in the end leads to their unlimited growth and survival. B cells are particulary prone to malignant transformation because processes that normally create imunoglobulin diversity, which is necessary for normal imunnologic response, often can cause chromosomal translocations and oncogenic mutations which include strong promoters of Ig genes, tumor–supressor genes, and oncogenes. Such aberations often lead to block in differentiation, prevention of apotosis and/or promotion of proliferation which can represent first oncogenic hit in B-cell transformation. In addition, most of B-cell lymphoma depend on expression of a BCR for their survival. In some lymphomas antigen activation of lyphoma cells through BCR signalling snd specific factors from the microenviroment are equally important in tumorogenesis. Reserach and development of funtional and structural genomics greatly improved understanding of mechanisms involved in B-cell lymphoma pathogenesis. Discovery of many signalling pathways which are aberrant in B cell lymphomas, leading to constitutive activation of many prosurvival pathways are now known. In addition, such research showed that B-cell lymhoma can be classified by their cell of origin because transformed cells often resemble normal stages of B-cell differentiation by immunophenotypic and genetic characteristics. In conclusion, development od field of genomics could lead to better understanding of mechanisms that drive B-cell lymphoma pathogenesis, all for one cause - efficient lymphoma therapies

Mechanisms of B-cell lymphoma development

Mehanizmi koji kontroliraju normalnu diferencijaciju i aktivaciju B-stanice često su u stanici limfoma kako bi u konačnici transformirana stanica imala neograničen rast i razvoj. B-stanice osobito su podložne malignim transformacijama zato što procesi koji dovode do velike raznolikosti imunoglobulina, koja je potrebna u normalnom imunološkom odgovoru, često vode do kromosomskih translokacija i onkogenih mutacija koje uključuju jake promotore Ig gena i mnoge tumor supresorske gene, kao i onkogene. Takvi događaji često vode do blokiranja diferencijacije, sprječavanja apoptoze i/ili poticanja proliferacije B-stanice koja konačno može biti prvi događaj u tranformaciji B-stanice u tumorsku stanicu. Dodatno, mnogi B-stanični limfomi trebaju ekspresiju BCR-a kako bi preživjeli, u nekim limfomima aktivacija stanice preko signalnog puta B-staničnog receptora te u nekima specifični faktori mikrookoliša tumora imaju važnu ulogu u tumorogenezi. Istraživanja na polju strukturne i funkcionalne genomike u zadnjih 10 godina značajno su doprinijela spoznajama o mehanizmima uključenim u patogenezu B-limfoma. Opisani su brojni signalni putovi koji su poremećeni u B-staničnim limfomima i koji vode do konsititutivne aktivacije brojnih drugih putova koji potiču preživljavanje tumorske stanice. Također, ovakav pristup istraživanja doveo je do klasifikacije B-limfoma prema stanicama od kojih su nastali budući da se zna kako su transformirane tumorske stanice u genetičkim i imunofenotipskim karakteristikama nalik nekom od tipova normalnih B-stanica. U godinama koje dolaze, nagli napredak genomike mogao bi dovesti do otkrića još mnogih drugih mehanizama koji su uključeni u transformaciju normalne B-stanice, što bi moglo pomoći u razvoju što uspješnijeg liječenja limfoma.Mechanisms that control normal B-cell differentiation and activation are frequently disrupted in lymphoma cells which in the end leads to their unlimited growth and survival. B cells are particulary prone to malignant transformation because processes that normally create imunoglobulin diversity, which is necessary for normal imunnologic response, often can cause chromosomal translocations and oncogenic mutations which include strong promoters of Ig genes, tumor–supressor genes, and oncogenes. Such aberations often lead to block in differentiation, prevention of apotosis and/or promotion of proliferation which can represent first oncogenic hit in B-cell transformation. In addition, most of B-cell lymphoma depend on expression of a BCR for their survival. In some lymphomas antigen activation of lyphoma cells through BCR signalling snd specific factors from the microenviroment are equally important in tumorogenesis. Reserach and development of funtional and structural genomics greatly improved understanding of mechanisms involved in B-cell lymphoma pathogenesis. Discovery of many signalling pathways which are aberrant in B cell lymphomas, leading to constitutive activation of many prosurvival pathways are now known. In addition, such research showed that B-cell lymhoma can be classified by their cell of origin because transformed cells often resemble normal stages of B-cell differentiation by immunophenotypic and genetic characteristics. In conclusion, development od field of genomics could lead to better understanding of mechanisms that drive B-cell lymphoma pathogenesis, all for one cause - efficient lymphoma therapies

Organisation of microtubules and force-balance in metaphase spindles of HeLa and Ptk1 cells

Tijekom stanične diobe, sestrinske kromatide razdvajaju se formiranjem metafaznog vretena, bipolarne molekularne makrostrukture samosastavljene od mikrotubula i pridruženih proteina. Trenutačno su u modelima metafaznog vretena opisane tri vrste mikrotubula: kinetohorni, interpolarni i astralni. U ovom radu analizirana su antiparalelna nekinetohorna vlakna mikrotubula koja se nalaze odmah ispod sestrinskih kinetohornih parova, a nazvana su premošćujuća vlakna. Primjenom fluorescentne mikroskopije živih stanica, pokazao sam da je ova struktura prisutna u različito obilježenim HeLa i Ptk1 stanicama, i to s različitim brojem mikrotubula u svakoj liniji. Također, analiziran je odgovor vanjskoga kinetohornog vlakna na lasersku ablaciju u staničnoj liniji Ptk1. Uočio sam zajedničku putanju kinetohornih vlakana, kinetohora i premoščujućih vlakana prema van što ukazuje na lateralnu povezanost između kinetohornih i premošćujućih vlakana. Također, pokazao sam da se udaljenost između kinetohora smanjuje nakon ablacije i da razina smanjivanja obrnuto korelira s udaljenošću mjesta ablacije od kinetohore. Dodatno, pokazao sam da se vanjski element vretena izravnava nakon ablacije, i to najbrže i najviše u liniji s najdebljim premošćujućim vlaknom, što ukazuje da su ta dva parametra korelirana. Konačno, usporedio sam neke od rezultata s predikcijama teorijskog modela, kako bih pokazao njegovu robusnost koristeći neke eksperimentalne podatke kao ulazne parametre. Dobiveni rezultati govore da su premošćujuća vlakna važna strukturalna komponenta koja ima ulogu u ravnoteži sila kompresije i tenzije u metafaznom vretenu.During cell division, sister chromatids are segregated by mitotic spindle, a bipolar self-assembly of microtubules and associated proteins. Current models of mitotic spindle recognize three distinct subpopulations of microtubules: kinetochore, interpolar and astral microtubules. The role of antiparallel non-kinetochore microtubule bundles, termed bridging fibers, positioned under sister kinetochores in HeLa and Ptk1 cell lines, was analysed in this study. Using live-fluorescent imaging analysis it was shown that these bundles are present in differently labelled HeLa and Ptk1 cell lines with distinct thicknesses. The response of outermost spindle element to laser ablation in Ptk1 cell line was also analysed. Joint outward movement of bridging and k-fibers was observed suggesting connection into single mechanical unit. Decrease in inter-kinetochore distance was also observed after ablation. That decrease is inversely correlated with distance of the cut from the kinetochore. In addition, spindle element straightening after ablation was observed in all cell lines but with strongest straightening in HeLa cell line with thickest bridging fiber, demonstrating that response to ablation and thickness of bridging fiber are correlated. Finally, some of obtained results were compared with theoretical model of the HeLa metaphase spindle confirming robustness of the model by using some of our experimental data as inputs. In conclusion, obtained results demonstrate that bridging fiber is important structural component of metaphase spindle involved in balancing compressive and tensile forces in spindle

Mechanisms of B-cell lymphoma development

Mehanizmi koji kontroliraju normalnu diferencijaciju i aktivaciju B-stanice često su u stanici limfoma kako bi u konačnici transformirana stanica imala neograničen rast i razvoj. B-stanice osobito su podložne malignim transformacijama zato što procesi koji dovode do velike raznolikosti imunoglobulina, koja je potrebna u normalnom imunološkom odgovoru, često vode do kromosomskih translokacija i onkogenih mutacija koje uključuju jake promotore Ig gena i mnoge tumor supresorske gene, kao i onkogene. Takvi događaji često vode do blokiranja diferencijacije, sprječavanja apoptoze i/ili poticanja proliferacije B-stanice koja konačno može biti prvi događaj u tranformaciji B-stanice u tumorsku stanicu. Dodatno, mnogi B-stanični limfomi trebaju ekspresiju BCR-a kako bi preživjeli, u nekim limfomima aktivacija stanice preko signalnog puta B-staničnog receptora te u nekima specifični faktori mikrookoliša tumora imaju važnu ulogu u tumorogenezi. Istraživanja na polju strukturne i funkcionalne genomike u zadnjih 10 godina značajno su doprinijela spoznajama o mehanizmima uključenim u patogenezu B-limfoma. Opisani su brojni signalni putovi koji su poremećeni u B-staničnim limfomima i koji vode do konsititutivne aktivacije brojnih drugih putova koji potiču preživljavanje tumorske stanice. Također, ovakav pristup istraživanja doveo je do klasifikacije B-limfoma prema stanicama od kojih su nastali budući da se zna kako su transformirane tumorske stanice u genetičkim i imunofenotipskim karakteristikama nalik nekom od tipova normalnih B-stanica. U godinama koje dolaze, nagli napredak genomike mogao bi dovesti do otkrića još mnogih drugih mehanizama koji su uključeni u transformaciju normalne B-stanice, što bi moglo pomoći u razvoju što uspješnijeg liječenja limfoma.Mechanisms that control normal B-cell differentiation and activation are frequently disrupted in lymphoma cells which in the end leads to their unlimited growth and survival. B cells are particulary prone to malignant transformation because processes that normally create imunoglobulin diversity, which is necessary for normal imunnologic response, often can cause chromosomal translocations and oncogenic mutations which include strong promoters of Ig genes, tumor–supressor genes, and oncogenes. Such aberations often lead to block in differentiation, prevention of apotosis and/or promotion of proliferation which can represent first oncogenic hit in B-cell transformation. In addition, most of B-cell lymphoma depend on expression of a BCR for their survival. In some lymphomas antigen activation of lyphoma cells through BCR signalling snd specific factors from the microenviroment are equally important in tumorogenesis. Reserach and development of funtional and structural genomics greatly improved understanding of mechanisms involved in B-cell lymphoma pathogenesis. Discovery of many signalling pathways which are aberrant in B cell lymphomas, leading to constitutive activation of many prosurvival pathways are now known. In addition, such research showed that B-cell lymhoma can be classified by their cell of origin because transformed cells often resemble normal stages of B-cell differentiation by immunophenotypic and genetic characteristics. In conclusion, development od field of genomics could lead to better understanding of mechanisms that drive B-cell lymphoma pathogenesis, all for one cause - efficient lymphoma therapies

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Background and Purpose: Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N- and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 O-linked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were detected, which demonstrates that plants are eligible candidates for production of recombinant therapeutics with fully humanized O- and N-glycans. Conclusion: Plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production

Polar Chromosomes - Challenges of a Risky Path

The process of chromosome congression and alignment is at the core of mitotic fidelity. In this review, we discuss distinct spatial routes that the chromosomes take to align during prometaphase, which are characterized by distinct biomolecular requirements. Peripheral polar chromosomes are an intriguing case as their alignment depends on the activity of kinetochore motors, polar ejection forces, and a transition from lateral to end-on attachments to microtubules, all of which can result in the delayed alignment of these chromosomes. Due to their undesirable position close to and often behind the spindle pole, these chromosomes may be particularly prone to the formation of erroneous kinetochore-microtubule interactions, such as merotelic attachments. To prevent such errors, the cell employs intricate mechanisms to preposition the spindle poles with respect to chromosomes, ensure the formation of end-on attachments in restricted spindle regions, repair faulty attachments by error correction mechanisms, and delay segregation by the spindle assembly checkpoint. Despite this protective machinery, there are several ways in which polar chromosomes can fail in alignment, mis-segregate, and lead to aneuploidy. In agreement with this, polar chromosomes are present in certain tumors and may even be involved in the process of tumorigenesis

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes

During cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes