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Većina istraživanja provedenih na miskantusu, provedena je na prirodnom sterilnom hibridu Miscanthus x giganteus. Nemogućnost generativne propagacije otežava veću komercijalnu primjenu. Zbog toga, kroz nekoliko europskih uzgojnih programa razvijeni su novi hibridi koji se trenutno intenzivno istražuju. Cilj ovog rada bio je odrediti energetska svojstva biomase novih hibrida roda Miscanthus, nakon čega je izračunat njihov ukupan potencijal. U istraživanju je korištena biomasa ukupno 13 novih hibrida roda Miscanthus, te M. giganteus kao kontrolni hibrid. Rezultati analiza upućuju na to da su svojstva biomase novih hibrida u skladu s energetski značajnim kulturama, pa tako i kontrolnim hibridom te da između njih postoje značajne razlike, ukoliko se uspoređuje ukupan energetski potencijal.

Melanoma is skin cancer developed from melanocytes and is considered to be one of the most aggressive tumor types due to high metastatic potential. One of the reasons why melanoma is prone to metastasis is a process known as a phenotype switching which is similar to epithelial–mesenchymal transition (EMT) since it includes EMT-related genes and signaling pathways. In addition, this process is shown to be implicated in resistance to targeted therapy. Depending on the BRAF mutation status, first line treatment options for melanoma can include targeted therapy or immunotherapy for BRAF-mutated melanoma, or immunotherapy for wild- type BRAF melanoma. Around 60% of melanoma carry BRAF mutation, of which almost 90% harbor BRAF V600E mutation that causes continuous activation of the MAPK signaling pathway. BRAF V600E mutated kinase can be targeted by BRAF inhibitor (BRAFi), vemurafenib, which is a first targeted therapy licensed for treatment of advanced melanoma. Although initial response in the clinic was very powerful, the disease relapses within several months of therapy introduction due to occurrence of acquired resistance to BRAFi therapy. Reactivation of the MAPK pathway occurs in the majority of BRAFi-resistant tumors, while the second most frequently activated signaling pathway is PI3K/AKT. So to investigate the underlying molecular mechanisms of BRAFi targeted therapy resistance in more detail, we generated and characterized two melanoma cell lines with acquired resistance to vemurafenib. The resistant cell lines exhibited specific features of slow cycling cells possibly as a consequence of vemurafenib-driven phenotype switching. These characteristics included morphological and molecular features of EMT-like cells, enhanced resistance to chemotherapy, changed levels of cell-cycle regulators and thus reduced proliferation. Additional features included decreased migration ability but tendency toward collective migration, and reactivation of MAPK or activation of PI3K/AKT signaling pathways, depending on the cell line. Reduced levels of NME1 and NME2 metastasis suppressor proteins were observed in primary vemurafenib-resistant cell line, which could originate from vemurafenib- acquired resistance and is one of the reasons for increased PI3K/AKT signaling. Further studies are required to reveal the vemurafenib-dependent suppressors of NME proteins, their association with PI3K/AKT signaling, and their impact on vemurafenib-resistant melanoma cell characteristics.

Organizacija je sastavni dio svakog sustava koji želi postići neki cilj. Organizacija je pojam koji svi intuitivno shvaćaju, međutim njegovo stvarno definiranje i opisivanje je vrlo složen postupak koji ne pruža jedinstveno rješenje. Na temelju analize organizacijske teorije te proučavanja Građevinskog fakulteta u Rijeci kao primjera organizacijske strukture, u diplomskom radu pod nazivom Organizacija javnog visokog učilišta u Republici Hrvatskoj na primjeru Građevinskog fakulteta u Rijeci, povezane su teorijske osnove s praktičnim primjerima. Dolazi se do spoznaje da ove teorije uistinu dobro opisuju stvarno stanje, ali i se ističu i neki problemi koji se javljaju kao posljedica ovakve organizacije. Organizacijska struktura je bitna odrednica efikasnosti organizacije. Struktura mora biti jasno definirana. Unutarnji i vanjski čimbenici strukture imaju presudnu ulogu u dizajniranju organizacije. Sustav visokog obrazovanja u RH ima vrlo formalno definiranu vertikalnu i horizontalnu strukturu. Upravo zbog formalne strukture visokoškolske ustanove se svrstavaju u birokratske organizacije koje su imaju formalnu strukturu. Promjena okoline zahtijeva promjenu organizacijskih struktura u cilju unaprjeđenja i opstanka organizacije.

In metastatic melanoma tumor suppressor protein p53 is not functional, although it is rarely mutated (less than 10%). Despite the low rate of mutations, p53 fails to function as tumor suppressor and tumor cells continue to proliferate and spread. Therefore, the question arises what in the metastatic melanoma prevents the wild type p53 protein to function. Since p53 interactions play a significant role in tumorigenesis, we have investigated whether the p53 protein function can be altered by interactions with p53 low molecular weight isoforms, p73 isoforms, and NME and GLI families of proteins. Therefore, we studied the protein expression profile of p53 and its potential interaction partners (p73/NME/GLI) in metastatic melanoma and in adjacent healthy skin tissue. In the study on 38 patients with metastatic melanoma, the expression of p53, p73, NME and GLI protein families was determined by western blot analysis. Protein expression analysis showed elevated expression of Δ133p53α, Δ160p53α, ΔNp73α, NME1, NME2, GLI1_160 kDa, GLI1_130 kDa and GLI2_133 kDa in tumor samples compared with healthy tissue samples. Strong correlations were detected only between TAp73β and p53α, and GLI3R and p53α. The remaining correlations were mostly moderate (p53α with Δ160p53α, TAp73α, ΔNp73α, GLI1_118 and GLI1_160). Interestingly, Δ133p53β was negatively correlated with p53α. We have shown that p53 forms complexes with p53 and p73 isoforms hence inhibiting its transcriptional and apoptotic activity. Although NME and GLI did not form complexes with p53, they were able to modify its activity. Defining the interactions between protein p53 and other proteins could contribute to better understanding of the molecular basis of melanoma and finally lead to the development of new approaches in melanoma treatment.

H. pylori infection causes damage of DNA in host cells, triggering the DNA damage response. Inhibition of DNA damage repair and p53 activity by H. pylori may be the links in the same chain of bacterial adaptive responses directed to prevent the host response to cellular stress. Unfortunately, this may have severe consequences for the host, increasing the risk of pathological changes and tumor development. Future studies of bacterial modulation of host stress responses will lead to better understanding of bacterial pathogenesis and help to identify potential targets in the prevention of cancers associated with bacterial infections.

Esophageal adenocarcinoma (EA) is one of the fastest rising tumors in the USA. The major risk factor for EA is gastroesophageal reflux disease (GERD). During GERD, esophageal cells are exposed to refluxate which contains gastric acid frequently mixed with duodenal bile. This may lead to mucosal injury and Barrett's metaplasia (BE) that are important factors contributing to development of EA. In this study, we investigated DNA damage in BE cells exposed to acidic bile salts and explored for potential protective strategies. Exposure of BE cells to acidic bile salts led to significant DNA damage, which in turn, was due to generation of reactive oxygen species (ROS). We found that acidic bile salts induce a rapid increase in superoxide radicals and hydrogen peroxide, which were determined using electron paramagnetic resonance spectroscopy and Amplex Red assay. Analyzing a panel of natural antioxidants, we identified apocynin to be the most effective in protecting esophageal cells from DNA damage induced by acidic bile salts. Mechanistic analyses showed that apocynin inhibited ROS generation and increases the DNA repair capacity of BE cells. We identified BRCA1 and p73 proteins as apocynin targets. Downregulation of p73 inhibited the protective effect of apocynin. Taken together, our results suggest potential application of natural compounds such as apocynin for prevention of reflux-induced DNA damage and GERD-associated tumorigenesis.

Nanoparticle-based therapies to treat cancer are widely evolving. Gold nanoparticles (GNPs) are small units that are inert and non-toxic to cells. The aim of this work was to study the radiosensitization effect of three types of GNPs to MDA-MB-231 breast cancer cells in vitro. Citrate, glutathione and aminodextran were used to modify GNPs surface and to increase their accumulation in cancer cells. All GNPs were well characterized with respect to their particle size, shape and colloidal stability in aqueous solution and cell media. Transmission electron microscopy demonstrated accumulation of GNPs in cytoplasmic vesicles, while the mass spectroscopy demonstrated the highest uptake of glutathione modified GNPs. GNPs alone show no significant effect on cell cycle demonstrated by flow cytometry and western blot analysis. As well, cells treated with GNPs in combination with x-ray irradiation show no significant effect on cell cycle. Nevertheless, long-term overall survival of cells treated with GNPs in combination with irradiation was reduced. In conclusion, we showed that GNPs have certain biological effect, but still the precise mechanism of their activity remains to be explored.

U normalnim fibroblastima čovjeka (NHF), fibroblastima koji eksprimiraju protein HPV E6 (NHF- E6) i staničnoj liniji osteosarkoma U2OS ispitali smo utjecaj pojačane ekspresije ΔNp73α na kontrolu staničnog ciklusa nakon oštećenja DNA. Pokazali smo da ΔNp73α dovodi do narušavanja kontrolne točke G2/M u stanicama U2OS uzrokujući povećanu učestalost poliploidije i doprinoseći genetskoj nestabilnosti. Učinak ΔNp73α u normalnim stanicama bio je umjereniji. Ovim radom pružili smo uvid u jedan od mogućih mehanizama kojima protein ΔNp73α ostvaruje svoje potencijalno onkogeno djelovanje.

Soft tissue sarcoma (STS) encompasses a plenitude of malignant tumors of the connective tissue. Most of these tumors are very invasive and the response to chemotherapy and radiation is very limited. The mechanism of soft tissue sarcoma tumorigenesis is still unclear ; therefore, they represent a major therapeutic challenge. Mutation of p53 is a common hallmark of cancer. It is mutated in more than 50% of human tumors. However, knowledge about the prognostic relevance of p53 mutations in STS is not comprehensive, and, according to our data, seems to be rare. Therefore, other mechanisms of p53 inactivation might be involved. As is already well accepted, p53 is expressed not only as full length, but also as isoforms, some of which inhibit wild-type p53. There are no studies that have examined the relative expression of p53 isoforms in STS so far. Here, we have analyzed the relative mRNA expression of the p53, Δ40p53, Δ133p53 (C- terminal isoforms), p53α, p53β and p53γ (N- terminal isoforms) in a panel of 5 STS cell lines, 26 STS specimens and 15 matched normal adjacent tissues using real-time PCR. Further we have analyzed their relationship to the outcome of disease. We have found that almost all isoforms were expressed equally, only p53γ was expressed at a higher level compared to p53. Further, we have observed that all three N-terminal isoforms are positively correlated to each other, compared to C-terminal ones which were not correlated. Additionally, higher p53β expression was significantly associated with longer overall survival (OS) of patients, while there was a trend toward significant association of higher Δ40p53 expression with longer OS. However, the higher Δ40p53 expression was positively correlated to reappearance of tumor with marginal statistical significance. These results have shown the implications of p53 isoforms in development of STS and might provide the elucidation for deregulated p53 function in STS.

The p53 activities are due, at least in part, to its ability to form oligomers that bind to specific DNA sequences and activate transcription. After discovering of p63 and p73, the p53 family members, it became evident that to understand the p53 pathway, all of them have to be taken into account. Since some mutant p53 proteins and ΔNp73 isoforms form heterocomplexes with TAp73, we asked whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73. Moreover, it has already been found that some isoforms form complex with wtp53 and some of them inhibit p53 tumor suppressor functions. Therefore, we studied the complex formation and co-immunoprecipitation assays show that all six p53 isoforms examined can form complexes with TAp73β and counteract TAp73β transactivation function but with different efficiency and in a promoter- dependent manner. Furthermore, apoptotic activity of TAp73β was augmented by coexpression of p53β, while Δ133p53α and β inhibit its apoptotic activity most efficiently. Additionally, we have wondered whether some mutant p53 can form complex with TAp73β and TAp63α and potentially act as dominant negative inhibitor. Coimmunoprecipitation assay has shown that some mutant p53 can form complex with TAp73β and TAp63α. Common polymorphism 72Arg binds more efficiently to p63 and p73 than the equivalent 72Pro. The results of reporter assays have shown that all mutant p53 inhibit transcriptional activity but with different efficiency. Furthermore, mutant p53248 and 280 are the most efficient inhibitors of TAp73β apoptotic activity of all tested mutant p53. Defining the interactions between p53/p63/p73 would gain insight into how the mutant p53 and p53 isoforms modulate the functions of p73 and p63 in tumorigenesis. Also deeper understanding of p53 protein network may guide decision on therapeutic approach and may provide prognostic information in cancer biology.

To understand the p53 pathways all three p53 family members have to be taken into account. p73 and p63 were shown to activate many p53 target genes, both are able to induce cell cycle arrest and apoptosis and are activated upon DNA damage. N-terminally truncated forms (ΔNp73/ ΔNp63) can act as potential transdominant inhibitors of TAp73, TAp63 (transactivation competent forms) and wild type p53. While TP73 and TP63 are rarely mutated in tumors, TP53 is mutated in many tumors losing its tumor suppressor functions, and often gaining oncogenic functions that stimulate tumor progression. Some of the mutant p53 oncogenic features are explained through interactions with other p53 family members, specifically tumor suppressor isoforms TAp63 and TAp73. Some mutants show strong binding to both TAp73β and TAp63α (R175H, L194F, R282W) while R280K weekly binds. Consequently, R280K does not inhibit transcriptional activity of TAp73β comparing to other mutants. It is established that p73 protein stability is predominantly regulated by Itch, HECT domain E3 ubiquitin ligase, which ubiquitinates p73 and targets it to proteosomal degradation. Since it was found that MDM2 functionally affects the relationship between p63/p73 and mutant p53, we assume that Itch could affect mutant p53/TAp73β and mutant p53/ TAp63α interaction. We have found that Itch inhibits the binding of all mutants to TAp73β what is the consequence of TAp73β proteasomal degradation. However, as was observed by western blot upon transfection of TAp73β and p53 mutants L194F and R280K, the expression of TAp73β was not affected by Itch. Deeper understanding of the interplay between Itch and mutant p53-p63/p73 may provide another new to target the activity of mutant p53.

Deregulation of the cell cycle progression is a common feature of tumor cells and one of the driving forces in tumorigenesis, mostly by leading to unscheduled proliferation and genomic instability. The canonical p53 protein (p53α) and its relative p73 are shown to have various roles in cell cycle regulation in stress conditions, especially as a part of DNA damage response signaling pathways. ΔNp73α, a potentially oncogenic isoform of p73 is often overexpressed in different human tumors and tumor cell lines in correlation with increased chemo- and radioresistance and worse prognosis. To understand better molecular mechanisms underlying the tumor-associated action of ΔNp73α, we investigated the effects of ΔNp73α ectopic expression on cycle progression of normal and tumor cells after DNA damage. Normal human fibroblasts (NHF) and U2OS human osteosarcoma cells infected with retroviral vectors harboring ΔNp73α were treated with different doses of γ-irradiation or topoisomerase II inhibitor ICRF-193 that specifically causes arrest in G2/M checkpoint. Cell cycle distribution was determined by FACS analysis measuring the DNA content after propidium iodide staining. The expression of several proteins involved in the control of cell cycle and DNA damage response was analysed by Western blot and immunofluorescence. U2OS cells overexpressing ΔNp73α showed higher percentage of tetraploid and polyploid cells after γ-irradiation in comparison to control cells, especially at longer time periods post- treatment. This was accompanied by lower p21, and higher cyclin B1 and A expression. This phenotype was even more pronounced upon treatment with ICRF-193. By contrast, the effect of ΔNp73α on DNA damage response in NHF was more modest, limited to certain increase of polyploidy. Our results show that ΔNp73α perturbs G2/M checkpoint control by abrogating cell cycle arrest after DNA damage leading to increased genomic instability. This could be one of the possible mechanisms of exerting its oncogenic role.

ΔNp73α represents a potentially oncogenic isoform of p73 protein. Its overexpression has been detected in various human tumors often correlating with increased chemoresistance and worse prognosis. Exploring ΔNp73α tumorigenic properties upon its overexpression revealed cell-type and condition-dependent roles. Our aim is to investigate the effects of ΔNp73α overexpression on cycle progression of normal and tumor cells and expression of different cell cycle regulators after DNA damage. Normal human fibroblasts (NHF) and U2OS human osteosarcoma cell line were infected with retrovirus carrying ΔNp73α gene. Cells were treated with different doses of γ-irradiation (5 and 10 Gy) or topoisomerase II inhibitor ICRF-193, and harvested at different time points. The expression of proteins involved in cell cycle regulation and DNA damage response (p21, p27, p53, pRB, cA, cB1, Chk1, Chk2) was determined by western blot. Distribution of cells in different cell cycle phases was determined using flow cytometer by measuring DNA content after propidium iodide staining. U2OS cells overexpressing ΔNp73α showed higher percentage of cells with 4N DNA content upon γ- irradiation compared to control cells, especially for longer time periods post-treatment (48 and 72h), and also increased number of cells with >4N DNA content (polyploidy). Significantly higher percentage of polyploid U2OS ΔNp73α overexpressing cells was also found after 48h of ICRF-193 treatment compared to control cells. The effect of ΔNp73α overexpression on cell-cycle distribution of NHF was weaker, possibly due to lower ectopic ΔNp73α levels compared to U2OS cells. Protein expression analysis of both cell types revealed various differences between control and cells overexpressing ΔNp73α upon both γ-irradiation and ICRF-193 treatment. ΔNp73α isoform was shown to influence cell response to different genotoxic treatments in U2OS, and to a lesser extent in NHF cells. Effects obtained in response to ICRF-193 and γ-irradiation suggest its potential role in G2/M DNA damage checkpoint, representing interesting basis for more detailed analysis of signalling pathways involved.

ΔNp73α represents a potentially oncogenic isoform of p73 protein. Its overexpression has been detected in various human tumors often correlating with increased chemoresistance and worse prognosis. Exploring its tumorigenic properties upon ΔNp73α overexpression revealed cell-type and condition-dependent roles. Our aim is to investigate the effects of ΔNp73α overexpression on cycle progression of normal and tumor cells and expression of different cell cycle regulators after DNA damage. Normal human fibroblasts (NHF) and U2OS human osteosarcoma cell line were infected with retroviruses carrying ΔNp73α gene. Cells were treated with different doses of γ-irradiation (5 and 10 Gy) or topoisomerase II inhibitor ICRF-193, and harvested at different time points. The expression of proteins involved in cell cycle regulation and DNA damage response (p21, p27, p53, pRB, cA, cB1, Chk1, Chk2) was detected by Western blot. Distribution of cells in different cell cycle phases was measured by FACS analysis by determining the DNA content after propidium iodide staining. U2OS cells overexpressing ΔNp73α showed higher percentage of cells with 4N DNA content upon γ- irradiation compared to control cells, especially at longer time periods post-treatment (48 and 72h), and also increased number of cells with >4N DNA content (polyploidy). Significantly higher percentage of polyploid U2OS ΔNp73α overexpressing cells was also found after 48h of ICRF-193 treatment compared to control cells. The effect of ΔNp73α overexpression on cell-cycle distribution of NHF was weaker, possibly due to lower ectopic ΔNp73α levels compared to U2OS cells. Protein expression analysis of both cell types revealed various differences between controls and cells overexpressing ΔNp73α after both treatments. ΔNp73α isoform was shown to influence cell response to different genotoxic treatments in U2OS, and to a lesser extent in NHF cells. Effects obtained in response to ICRF-193 and γ-irradiation suggest its potential role in G2/M DNA damage checkpoint, representing interesting basis for more detailed analysis of signalling pathways involved.

The p53 activities are due, at least in part, to its ability to form oligomers that bind to specific DNA sequences and activate transcription. Since some mutant p53 proteins and ΔNp73 isoforms form heterocomplexes with TAp73, we asked whether p53 isoforms can do the same and potentially act as dominant- negative inhibitors of TAp73. Moreover, it has already been found that some isoforms form complex with wtp53 and some of them inhibit p53 tumor-suppressor functions. Therefore, we studied the complex formation and co- immunoprecipitation assays show that all six p53 isoforms examined can form complexes with TAp73β, whereas only Δ133p53α/β/γ isoforms form complex with TAp73α. All p53 isoforms counteract TAp73β transactivation function but with different efficiency and in a promoter- dependent manner. Furthermore, apoptotic activity of TAp73β was augmented by coexpression of p53β, whereas Δ133p53α and β inhibit its apoptotic activity most efficiently. We have determined the half-life of different p53 isoforms: p53γ isoform has the shortest half-life, whereas Δ133p53γ has the longest half-life. Inhibitory interactions of two proteins in complex often lead to their stabilization. However, only three isoforms (Δ133p53α, Δ133p53β and Δ40p53α) stabilize TAp73β. We are convinced that defining the interactions between p53/p73 would give a new insight into how the p53 isoforms modulate the p73 functions in tumorigenesis.

Introduction. The p53 activities are due, at least in part, to its ability to form oligomers that bind to specific DNA sequences and activate transcription. Since some mutant p53 proteins, as well as ΔNp73 isoforms form heterocomplex with TAp73, we asked whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73. Moreover, it was already found that some isoforms form complex with wtp53 and some of them inhibit p53 tumor suppressor functions. Material and method. The p53 null human cell line H1299 cells were used as a model. Proteins were extracted and western blot was preformed by standard methods. The complex formation was determined by coimmunoprecipitation assay. Transcriptional and apoptotic activity TAp73β were measured by Dual-Glo Luciferase Assay or Annexin- V-FLUOS staining kit, respectively. The half lives of different p53 isoforms have been determined using pulse chase method with cyclohexamide. Results and discussion. Coimmunoprecipitation assay has shown that all six p53 isoforms examined can form complexes with TAp73β, while only D133p53 isoforms (α, β and γ) with TAp73α. All p53 isoforms counteracts TAp73 transactivation function but with different efficiency and in a promoter- dependent manner. Furthermore, apoptotic activity of TAp73β was augmented by coexpression of p53, while Δ133p53α and Δ133p53β inhibit its apoptotic activity most efficiently. The half lives of different p53 isoforms have shown that p53γ isoform has the shortest while Δ133p53γ has the longest half life. Inhibitory interactions of two proteins in complex often lead to their stabilization. However, only three isoforms (Δ133p53α, Δ133p53β and Δ40p53α) stabilize TAp73β. Conclusion. p53 isoforms can modulate p53 family tumor suppressor activities, and the expression ratio between members of the family is probably the most important factor in determining the cell fate and outcome.

p73 gene encodes numerous protein isoforms divided into two groups according to alternative promoter usage: tumor-suppressive TAp73 isoforms whose functions partly overlap with those of wild-type p53, and potentially oncogenic ΔNp73 isoforms which inhibit wild-type p53, p63 and p73. Since ΔNp73 isoforms are often overexpressed in human tumors showing enhanced chemoresistance, they are considered as potential markers of worse prognosis. Our investigation is focused on the impact of ΔNp73α overexpression on cell cycle regulation and response to different DNA damaging agents (ICRF-193, bleomycin, γ-irradiation) in normal and tumor human cells. ΔNp73α was overexpressed in wild-type human dermal fibroblasts (HDF-WT), HDF expressing human papilloma virus HPV16-E6 oncoprotein (HDF-E6), and U2OS human osteosarcoma cell line by retroviral infection. Our video-microscopy experiments showed that in HDF-WT ΔNp73α overexpression abrogates G2 cell cycle arrest after treatment with topoisomerase II inhibitor ICRF-193. In agreement with this, we found lower p21 expression in cells with ΔNp73α compared to control ones upon ICRF-193 treatment. To explore potential role of ΔNp73α at the G2/M checkpoint, HDF-WT and HDF-E6 were synchronized at G1/S boundary and exposed to γ- irradiation (12 Gy) after release, but FACS analysis showed no significant difference between cells expressing ΔNp73α and control. FACS analysis of γ-irradiated (3 and 5 Gy) HDF-WT and U2OS cells revealed higher percentage of polyploidy in cells overexpressing ΔNp73α. Further analysis of cell cycle progression and specific DNA damage signaling pathways will help to elucidate the role of ΔNp73α in the process of oncogenesis.

No data has been reported on Smads mediated activation events in fibrosarcoma cells. To analyse the activation status of U4C fibrosarcoma cells upon transfection with different Smads constructs we have followed immunoreactivity of Smad-4 molecule in the nucleus by confocal microscopy. Thus, wild type U4C cells, cells deficient in Jak-1 molecule and overexpressing it have been transfected with Smad-1, Smad-2 or Smad 3 in order to see which signaling pathway - TGF- ( Smad-2 and Smad-3 mediated) or BMPs ( Smad-1 mediated) eventually become active We have observed high constitutive immunoreactivity of Smad-4 molecule in the nucleus of Jak-1 transgenic cells. This immunoreactivity has been reduced upon transfection with Smad 1, Smad-2 or Smad- 3. Also, the same cells have been stained with DAPI to evaluate apoptosis. Since NF-kappa B transcription factor has a dual role - pro and anti-apoptotic, DAPI stained cells were analysed for immunoreactivity of p65/NF-B subunit in the nucleus, a marker of NF-B activation. p65 subunit has been chosen to follow the translocation in to nucleus of both p52/p65 and p50/p65 NF-kappaB complexes. Western blot analysis revealed that both, wild type and genetically modified U4C cells, express p65, though, in genetically modified cells at slightly higher level.

p73 exists in multiple isoforms which could be divided into two groups: one containing transactivation domain (TA) and another amino- terminally truncated (ΔN) isoforms. While TAp73 isoforms show tumor-suppressive functions similar to those of wild-type p53, ΔNp73 isoforms inhibit wild-type p53 as well as TAp63 and TAp73, and are considered as potential oncogenes. ΔNp73 isoforms are overexpressed in many tumors correlating with enhanced chemoresistance and poorer disease outcome. The aim of our work is to investigate how ΔNp73α overexpression (OE) affects cell cycle regulation and DNA damage response in normal human cells. To this end we infected wild-type human dermal fibroblasts (HDF-WT) as well as HDF expressing human papilloma virus HPV16-E6 oncoprotein (HDF- E6), which promotes p53 degradation, with retroviral vectors carrying ΔNp73α gene. Preliminary results show that HDF cells overexpressing ΔNp73α are more prone to entry into mitosis compared to control ones after treatment with ICRF-193 which causes G2 arrest or delay, obtained by video-microscopy. In accordance with this result, the lower expression of p21 is observed in cells with ΔNp73α compared to control ones after ICRF-193 treatment. In order to investigate whether ΔNp73α has specific role at the G2/M checkpoint, HDF and HDF E6 cells were synchronized by hydroxyurea in G1/S. Released cells were γ-irradiated (12 Gy) and analysed by FACS. As no significant difference could be seen between cells expressing ΔNp73α and control cells, detailed analysis of the number of cells that manage to enter mitosis needs to be taken. Further investigation could contribute to understanding the mechanisms by which ΔNp73 isoforms carry out its oncogenic function in normal cells, shedding new light on the process of tumorigenesis in general.

After discovering of p63 and p73, it became evident that to understand the p53 pathways, all of family members have to be taken into account. p53 tumor suppressor protein is crucial in the cell growth control and the maintenance of genomic stability. These activities are due, at least in part, to its ability to form tetramers that bind to specific DNA sequences and activate transcription. The homologues of p53, proteins p63 and p73, can transcriptionally activate p53 target genes in vivo. Both p63 and p73 generate transactivating forms (TAp73/TAp63) as well as a number of N-terminally truncated transactivation-deficient transdominant isoforms (called ΔTAp73/ΔTAp63). It was recently discovered that p53, like p73, has a second promoter P2 and undergoes alternative splicing to generate multiple isoforms that might play important roles in carcinogenesis. Since some mutant p53 proteins form complexes with TAp73α or TAp73β it was important to find out whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73 and TAp63. All six p53 isoforms can form complex with TAp73β, while only isoforms D133p53, D133p53β and D133p53γ can form complex with TAp73α. Inhibitory interactions of two proteins in complex often lead to their stabilization. Our results have shown that only three isoforms (Δ133p53, Δ133p53β and Δ40p53) stabilize TAp73β. Furthermore, we have shown that all isoforms of p53 inhibit transcriptional activity but with different efficiency. The apoptotic acitivity of TAp73β was augmented by coexpression of p53, but Δ133p53 and Δ133p53β inhibit its apoptotic activity most efficiently. The half lives of different p53 isoforms were determined - p53γ isoform has the shortest, while Δ133p53γ has the longest half life. Defining the interactions between p53 family members would gain insight into how the p53 isoforms modulate the functions of p73. The discovery of p53/p73 network could have a major clinical impact in prognostic use and p53 targeted drug design.

After recent discovering of p63 and p73, the p53 family members, it became evident that to understand the p53 pathways, all of them have to be taken into account. p73 was shown to activate many p53 target genes, it is able to induce cell cycle arrest and apoptosis and is activated upon DNA damage. N-terminally truncated forms (ΔNp73) can act as potential transdominant inhibitors of both TAp73 (transactivation competent form) and wild type p53. On the other hand, mutant p53 proteins, most of which have lost tumor suppressor activity and gained oncogenic potential, are dominant negative inhibitors of TAp73. The mechanism of transdominant inhibitory activity can be explained through physical interaction. To determine the effect of tumor-derived p53 mutants (R175H, R248W, L194F, R249S, R280K, R282W) on p73 transcriptional activity, we performed reporter assay. The results have shown that all mutant inhibit transcriptional activity but with different efficiency. Furthermore, we have shown that p53 R248W and R280K mutants are the most efficient inhibitors of TAp73β apoptotic activity. Inhibitory interactions of two proteins often lead to stabilization of their components. We tested whether mutant p53 proteins can also induce stabilization and concomitant inactivation of p73 proteins. Our results have shown that all mutant p53 stabilize TAp73β with similar effect. Defining the interactions between p53/p73 would gain insight into how the p53 modulate the functions of p73. The discovery of p53/p73 network could have a major clinical impact in prognostic use and p53 targeted drug design.

Tumor supresorski gen p53 ima kritičnu ulogu u kontroli staničnog rasta i održavanju genetske stabilnosti. Ove aktivnosti posljedica su činjenice da p53 stvara tetramere koji se vežu za određeni slijed na DNA i aktiviraju transkripciju. Gubitkom tumor supresorske aktivnosti, mutirani proteini p53 dobivaju onkogeni potencijal što rezultira nekontroliranim rastom stanica tumora. Otkrivena su i dva srodnika gena p53- p63 i p73, koji pokazuju veliku strukturnu sličnost s genom p53, ali funkcionalno velike razlike. Sva tri gena imaju dva promotora, što zajedno s alternativnim izrezivanjem i alternativnim početkom translacije daje brojne izoforme proteina s različitim funkcijama. Tako geni p63 i p73 imaju vrlo složenu strukturu i kodiraju dvije skupine izoformi proteina koje imaju suprotno funkcionalno djelovanje- izoforme s transaktivacijskim područjem (TAp63/73) koje imaju tumor supresorsko djelovanje i izoforme bez transaktivacijskog područja (ΔNp63/73) s potencijalnim onkogenim djelovanjem djeluju kao transdominantni inhibitori TAp73 i divljeg tipa p53. Nedavno je utvrđeno da i p53 sadrži još jedan promotor, te da dolazi do sinteze višestrukih izoformi, čija funkcija nije još u potpunosti jasna. U ovom preglednom radu opisane su različite izoforme p63, p73 i p53 te njihova uloga u razvoju i tumorigenezi. Istraživanja članova obitelji p53, posebice njihovih međusobnih interakcija pridonijet će rasvjetljavanju uloge ovih proteina u nastanku i razvoju tumora.

TP53 tumor suppressor protein is crucial in the cell growth control and the maintenance of genomic stability. These activities are due, at least in part, to its ability to form tetramers that bind to specific DNA sequences and activate transcription. The homologues of p53, proteins p63 and p73, can transcriptionally activate p53 target genes in vivo. Both p63 and p73 generate transactivating forms (TAp73/TAp63) as well as a number of N-terminally truncated transactivation-deficient transdominant isoforms (called ΔTAp73/ΔTAp63). It was recently discovered that p53, like p73, has a second promoter P2 and undergoes alternative splicing to generate multiple isoforms that might play important roles in carcinogenesis. Since some mutant p53 form complexes with TAp73α or TAp73β it was important to find out whether p53 isoforms can do the same and potentially act as dominant-negative inhibitors of TAp73 and TAp63. All six p53 isoforms can form complex with TAp73β, while only isoforms D133p53, D133p53β and D133p53γ can form complex with TAp73α. Inhibitory interactions of two proteins in complex often lead to their stabilization. Our results have shown that only three isoforms (Δ133p53, Δ133p53β and Δ40p53) stabilize TAp73β. Furthermore, we have shown that all isoforms of p53 inhibit transcriptional activity but with different efficiency. The apoptotic acitivity of TAp73β was augmented by coexpression of p53, but Δ133p53 and Δ133p53β inhibit its apoptotic activity most efficiently. The half lives of different p53 isoforms were determined - p53γ isoform has the shortest, while Δ133p53γ has the longest half life. We have found additional p53 splice variants (p53ζ, p53δ and p53ε) arising from alternative splicing of exon 6 and intron 9, respectively, present in 52.9% ovarian cancer cell lines and in 54, 7% primary ovarian cancers. The interaction between these p53 variants and p73 has to be determined. Defining the interactions between p53/p73 would gain insight into how the p53 isoforms modulate the functions of p73. The discovery of p53/p73 network could have a major clinical impact in prognostic use and p53 targeted drug design.

Stanična senescencija je trajni izlazak stanica iz ciklusa do kojeg dolazi zbog skraćivanja telomera, aktivacije onkogena i oštećenja DNA. Protein p73 (član obitelji proteina p53) ima niz uloga u odgovoru na oštećenje DNA, a njegova uloga u tumorigenezi je složena. Postoje mnogobrojne izoforme proteina p73 svrstane u dvije glavne skupine: TAp73 i ΔNp73. Potaknuti potencijalnim onkogenim djelovanjem izoforme ΔNp73α ispitali smo njezin utjecaj na ulazak stanica u senescenciju izazvanu oštećenjem DNA γ-zračenjem u normalnim fibroblastima i staničnoj liniji osteosarkoma čovjeka. Senescentne stanice detektirane su praćenjem endogene β-galaktozidazne aktivnosti povezane sa starenjem, pri čemu je pojačana ekspresija ΔNp73α dovela do umjerenog porasta udjela senescentnih stanica. Metodom Western hibridizacije ispitana je ekspresija proteina uključenih u regulaciju staničnog ciklusa nakon oštećenja DNA, a dobiveni rezultati ukazuju na višu ekspresiju markera stanične proliferacije pod utjecajem ΔNp73α. Dobiveni rezultati predstavljaju vrijednu podlogu za buduća istraživanja uloge izoforme ΔNp73α u tumorigenezi. Cellular senescence is permanent cell cycle exit caused by telomere shortening, oncogene activation and DNA damage. P73 protein (a p53 protein family member) has different functions in DNA damage response, and its role in tumorigenesis is complex. There are multiple p73 protein isoforms divided into two main groups: TAp73 and ΔNp73. Encouraged by potentially oncogenic activity of ΔNp73α isoform, we investigated its impact on senescence onset after DNA damage induced by γ-irradiation in normal human fibroblasts and osteosarcoma cell line. Senescent cells were detected by analysis of endogenous senescence associated β-galactosidase activity, showing moderate increase in the frequency of senescence in cells overexpressing ΔNp73α. Expression of proteins involved in cell cycle regulation after DNA damage was analyzed by Western blot, suggesting higher expression of the markers of cellular proliferation caused by overexpression of ΔNp73α. These results represent a valuable basis for future investigation of the ΔNp73α role in tumorigenesis.