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Protein p16, kodiran genom INK4A, pripada obitelji gena INK4 koja ima ulogu tumor supresora i negativno regulira signalni put pRb-E2F. Genetskim promjenama gena INK4A dolazi do potpune inaktivacije ili do smanjene funkcije proteina p16. Karcinomi glave i vrata su tumori koji nastaju na području usne šupljine, ždrijela, grkljana i žlijezda slinovnica. U 50-70% karcinoma glave i vrata nađena je inaktivacija gena INK4A. U ovom diplomskom radu istraživao sam utjecaj ekspresije proteina p16 u stanicama tumora glave i vrata A253, FaDU i Detroit 562, na ekspresiju ciljnih gena te modulaciju tretmana s kemoterapeuticima. Ustanovio sam da kemoterapeutici induciraju ekspresiju gena INK4A u nekim staničnim linijama te da dodatni tretman s p16 pojačava taj efekt. Prateći ekspresiju ciljnih gena p16 utvrdio sam da se genska i proteinska ekspresija gena BIRC5 (proteina survivin) smanjuje pod utjecajem p16. Genska ekspresija gena BIRC5 se smanjivala nakon tretmanima kemoterapeuticima. Tretman doksorubicinom povećao je gensku ekspresiju E2F1 nekim linijama, dok ju je tretman etopozidom i p16 smanjio. Tretmani kemoterapeuticima i p16 utjecali su na stanični ciklus, uzrokovali oštećenje DNA, te apoptozu i smrt stanica. Kombinirani tretmani s p16 i kemoterapeuticima nisu pokazivali jednoznačne rezultate u staničnim linijama, pa se nije moglo zaključiti ima li p16 ikakav dodatni utjecaj. P16 is a protein coded by the INK4A gene that belongs to INK4 gene family. They have a tumor suppressor role and negatively regulate the pRb-E2F signal pathway. When a genetic mutation appears in INK4A, it leads to inactivation of the protein p16 or its improper function. Head and neck carcinomas can occur in the oral cavity, pharynx, larynx or in the salivary glands. In 50-70% of head and neck cancers the inactivation of INK4A was found. In this graduation thesis I studied the influence of p16 protein expression in head and neck cell lines A253, FaDU and Detroit 562 on the expression of target genes and its effect of the treatment with chemotherapeutic drugs. I found that chemotherapeutics induce expression of INK4A gene in some cell lines and that additional treatment with p16 amplifies that effect. Following the expression of the p16 target genes I found that the protein and gene expression of BIRC5 gene (survivin protein) is lowered under the influence of p16. Gene expression of the BIRC5 gene was lowered with chemotherapeutics treatment. Treatment with doxorubicin increased the E2F1 gene expression in some cell lines, but the treatment with etoposide and p16 lowered E2F1 expression. Treatment with chemotherapeutics and p16 influenced the cell cycle, DNA damage, apoptosis, and cell death. Combined treatment of p16 and chemotherapeutics did not show consistent results in cell lines, so I could not make a conclusion if p16 has any additional influence.

Jezgrica ima značajnu ulogu u sintezi ribosoma te je važan senzor stresa u stanici. Sinteza ribosoma neophodna je za povećanje kapaciteta sinteze proteina tijekom rasta, diobe i diferencijacije stanica. Poznato je da su jezgrice povećane u većini zloćudnih tumora zbog intenzivne sinteze ribosoma koja je potrebna za brzi rast i diobu stanica. Kao posljedica povećane sinteze ribosoma često dolazi do morfoloških promjena struktura u jezgrici. Brojni protutumorski lijekovi svoj učinak ostvaruju putem inhibicije sinteze ribosoma na različitim koracima i inhibicije rasta stanice te na taj način onemogućuju rast i diobu zloćudno preobraženih stanica. Za bolje razumijevanje molekularnih mehanizama putem kojih protutumorski lijekovi, etopozid i doksorubicin, ostvaruju svoj učinak u liječenju zloćudnih tumora u ljudi, u humanim stanicama A549 ispitala sam njihov utjecaj na aktivaciju supresora tumora p53 upotrebom metode Western blot. Osim toga, ispitala sam njihov učinak na morfološka svojstva jezgrice određivanjem izražaja biljega UBF-a i fibrilarina upotrebom metode imunofluorescencije te njihov utjecaj na aktivnost RNK polimeraze I određivanjem izražaja glasničke RNK za 47S pre-rRNK metodom qRT-PCR. Tretman stanica s etopozidom i doksorubicinom doveo je do aktivacije supresora tumora p53 kao i aktivacije njegovog ciljnog gena, p21. Tretman stanica s doksorubicinom doveo je do značajnih morfoloških promjena jezgrice koje se očituju u formiranju nakupina fibrilarina i UBF-a na rubovima oštećene jezgrice, dok tretman stanica s etopozidom nije imao utjecaj na morfološka svojstva jezgrice. Osim toga, doksorubicin je spriječio izražaj 47S pre-rRNK što je rezultiralo inhibicijom sinteze ribosoma, dok etopozid nije imao utjecaj na izražaj 47S pre-rRNK. Poznavanje molekularnih mehanizama djelovanja protutumorskih lijekova ima važnu ulogu kod izbora terapije za liječenje. The nucleolus has a significant role in ribosome biogenesis and is an important sensor of stress in the cell. Ribosome biogenesis is necessary to increase protein synthesis capacity during cell growth, cell division and differentiation. It is known that in most malignant tumors, nucleolus has been increased due to the intense ribosome biogenesis which is needed for rapid growth and cell division. As a result of increased ribosome biogenesis, morphological changes in the structure of the nucleus often occur. The effects of numerous anti-inflammatory drugs are achieved by inhibition of ribosomal biogenesis at different steps and inhibition of cell growth thus prevent the growth and division of malignant cells. For a better understanding of the molecular mechanisms by which anti-inflammatory drugs, etoposide and doxorubicin, have an effect on the treatment of malignant tumors in humans, in human A549 cells I tested their influence on the activation of p53 tumor suppressors using the Western blot method. In addition, I tested their effect on the morphological properties of nucleolus by determining expression of UBF and fibrillarin using the immunofluorescence method. Also I tested their influence on the activity of RNA polymerase I and the expression of the messenger RNA for 47S pre-rRNA using the qRT-PCR method. The treatment of cells with etoposide and doxorubicin resulted in activation of p53 tumor suppressor as well as activation of its target gene, p21. Also, treatment with doxorubicin resulted in significant morphological changes in the nucleolus manifested in the formation of fibrillarin and UBF clots at the edges of the damaged nucleolus, while the treatment with etoposide had no effect on the morphological properties of the nucleolus. In addition, doxorubicin prevented the expression of 47S pre-rRNA resulting in inhibition of ribosome biogenesis, while etoposide had no effect on the expression 47S pre-rRNA. Understanding the molecular mechanisms of anti-inflammatory drugs plays an important role in selecting treatment therapy.

Karcinom pluća je najčešći karcinom u oba spola. Glavni uzrok u većini slučajeva karcinoma pluća jest konzumacija duhanskih proizvoda. Mali dio karcinoma pluća uzrokuju tvari na koje se udišu pri radu, kao što su azbest i nikal. SCLC čini 20% karcinoma pluća. Pacijenti sa SCLC-om se liječe primarno kemoterapijom, jer je SCLC dobro osjetljiv na kemoterapiju i jer je SCLC najčešće već u vrijeme dijagnoze proširen po organizmu. SCLC se dijeli na ograničeni i prošireni stadij. Ograničeni stadij jest kad je karcinom lokaliziran u jednom hemitoraksu, a prošireni stadij označava bolest kad je karcinom proširen izvan jednog hemitoraksa. Kirurgija ne igra važnu ulogu u liječenju jer se većina SCLC-a otkrije u proširenom stadiju. U pacijenata s ograničenim stadijem bolesti, uz kemoterapiju se provodi istovremena radioterapija, dok se kod pacijenata sa proširenim stadijem provodi samo kemoterapija. Najčešće korištena kombinacija citotoksičnih lijekova je etopozid i cistplatin. Kemoterapija se provodi između 4 do 6 ciklusa. SCLC ima sklonost širenja u mozak, pa se kod većine pacijenata provodi profilaktičko zračenje mozga s ciljem sprečavanja metastaza u mozgu. U većini pacijenata nakon nekog vremena pojavi se recidiv SCLC. Topotekan je izbor lijeka u liječenju recidiva SCLC. Prognoza je vrlo loša, stopa petogodišnjeg preživljenja kod ograničenog stadija je 20 mjeseci, a kod pacijenata s proširenim stadijem, između 7 i 11 mjeseci. Lung cancer is the most common cancer in men and women. The main cause of most cases of lung cancer is the consumption of tobacco products. A small fraction of lung cancer is caused by substances that are inhaled during work, such as asbestos and nickel. SCLC accounts for 20% of lung cancer. Primary, patients with SCLC are treated with chemotherapy because it is well-sensitive to chemotherapy and at the time of diagnosis SCLC is usually augmented through the body. SCLC is divided into limited-stage and extensive-stage. Limited stage SCLC is confined to one hemithorax and extended stage in SCLC is extended beyond one hemithorax. Surgery doesn't have an important role in the treatment of SCLC because most times SCLC is detected in the extended stage. In patients with limited-stage disease, chemotherapy with radiation therapy is performed simultaneously, while in patients with an extended stage only chemotherapy is carried out. The most commonly used combination of cytotoxic drugs is etoposide and cistplatin. Chemotherapy is carried out between 4 to 6 cycles. SCLC tends to spread to the brain, so most patients get prophylactic brain radiation to prevent metastases in the brain. In most patients a relapse of SCLC appears. Topotecan is the drug of choice in the treatment of relapse SCLC. The prognosis is very poor, the survival rate in those with the limited stage is 20 months, and in patients with extended stage, it's between 7 and 11 months.