Search

Zloćudni tumori nastaju zbog poremećaja regulacijskih mehanizama, neophodnih za rast i dijeljenje stanice. Nerijetko su im u podlozi nastanka mutacije u različitim genima, najčešće protoonkogenima i tumorsupresorskim genima. Početak zloćudne preobrazbe povezan je s nastankom klona brzoproliferirajućih stanica, na koji se nastavljaju evolucija i selekcija klonova s najsnažnijim replikacijskim potencijalom. Zloćudni tumori posjeduju deset temeljnih obilježja, među kojima je i reprogramirani metabolizam. Metabolička aktivnost stanica zloćudnog tumora promijenjena je u odnosu na zdrave stanice od kojih je tumor nastao, a naročito se intenzivno istražuje u odnosu na metabolizam glukoze i glutamina. Za razliku od zdravih stanica koje svoje metaboličke puteve preusmjeravaju u glikolizu u anaerobnim uvjetima, stanice zloćudnih tumora sklone su metaboliziranju velikih količina glukoze glikolizom u uvjetima u kojima je opskrba kisikom optimalna. Ovaj se fenomen naziva aerobna glikoliza ili Warburgov učinak. Osim glukoze, stanice zloćudnih tumora imaju i veliku potrebu za glutaminom, koji služi kao izvor dušika u biosintetskim reakcijama. Biosinteza i iskorištavanje serina u metabolički reprogramiranim stanicama smatra se iznimno važnim za njihovu proliferaciju. Prvi enzim biosinteze serina je fosfoglicerat dehidrogenaza (PHGDH), čija je povećana ispoljenost, kao posljedica amplifikacije dijela kromosoma, dokazana u nekim vrstama zloćudnih tumora. Cilj ovog istraživanja bio je provjeriti postojanje stanično specifične ispoljenosti PHGDH na razini transkripta (RT-qPCR), proteina i u odnosu na unutarstanični smještaj proteina (Western blot), u odgovoru na dvije vrste gladovanja, u tri linije stanica podrijetlom od tumora glave i vrata, te debelog crijeva. Rezultati pokazuju da se ispoljavanje ciljnog proteina u sve tri linije stanica pojačava u gladovanju. Ova promjena može (FaDu), i ne mora (Detrot562, HT29) biti praćena povećanjem transkripcijske aktivnost gena PHGDH. Neočekivano je otkriveno da u linijama stanica podrijetlom od tumora glave i vrata (FaDu, Detroit562), PHGDH u gladovanju ulazi u jezgru, dok ovaj fenomen izostaje u stanicama podrijetlom od karcinoma debelog crijeva (HT29)., The occurrence of malignant tumors is consequential to disturbed regulatory mechanisms that are needed for cellular growth and division. Mutations of protooncogenes and tumor suppressor genes are frequently associated with their genetic background. Initiation of malignant transformation relates to the onset of a highly proliferative clone, while its evolution depends on the selection of clones with the most prominent replicative potential. Reprogrammed metabolism is one of ten basic cancer hallmarks. Metabolic activity of cancer cells is changed with respect to the cells from which specific cancer originates. Currently, the most intense research efforts are focused on the metabolism of glucose and glutamine. Contrary to untransformed cells which utilize glycolytic pathways when deprived of oxygen, malignant cells utilize a high amount of glucose through the same pathway, even when sufficient oxygen is available. This phenomenon is known as aerobic glycolyisis or the Warburg effect. In addition to glucose, malignant cells need glutamine, which they use as a source of nitrogen in biosynthetic reactions. Serine metabolism is considered to be of utmost importance for the proliferation of malignant cells. The first enzyme of serine biosynthesis is phosphoglycerate dehydrogenase (PHGDH). This enzyme is highly expressed in some malignant tumors, as a consequence of chromosome amplification. The objective of this research was to explore the cellular specific expression of PHGDH at the level of the gene transcriptional activity (RT-qPCR), and the level and localization of the protein product, respectively, in three cell lines originating from head and neck malignant tumors and colon carcinoma, exposed to two types of starvation. In all three cell lines, exposure to starvation is associated with an increased level of the PHGDH protein. This change may (FaDu) and may not (Detroit562, HT29) be related to transcriptional activity of the PHGDH gene. During starvation, PHGDH unexpectedly translocates into the nucleus of the head and neck originating cell lines (FaDu, Detroit562), while the effect is absent in colon cancer originating HT29 cells.

Protein p53 kao tumor supresorima središnju ulogu u kontroli staničnog ciklusa i tumorigeneze. Nedavno je otkriveno da gen p53 kodira 9 izoformi (p53, p53 β , p53 γ , 133p53, 133p53 β , 133p53 γ , 40p53, 40p53 β i 40p53 γ ) nastalih alternativnim izrezivanjem ili prepisivanjem s alternativnog promotora u intronu 4. Ekspresija izoformi tkivno je specifična, a biološka funkcija im nije u potpunosti razjašnjena. Gen p53 ima dva srodnika velike strukturne i funkcionalne sličnosti, p63 i p73 . Spomenuti geni također imaju određen broj izoformi, koje djelovanje proteina p53 čine još složenijim. Testom luciferaze istražena je transkripcijska aktivnost izoformi p73 β , p53, p53 β , p53 γ , 133p53, 133p53 β , 133p53 γ i 40p53 transfeciranih u stanice H1299, koje nemaju eksprimiran protein p53 divljeg tipa. Dokazana je transkripcijska aktivnost svih istraživanih izoformi. Izoforme p73 β i p53 β pokazuju najveću transkripcijsku aktivnost. Izoforme p53 γ i 40p53 pokazuju značajnu razinu transkripcijske aktivnosti, dok izoforme 133p53, 133p53 β i 133p53 γ imaju izrazito nisku razinu transkripcijske aktivnosti. Metodom Western blot dokazana je ekspresija svih istraživanih izoformi na razini proteina. Protein p53 as a tumor suppressor plays a central role in the cell cycle control and tumorigenesis. It was recently discovered that the gene p53 encodes nine different protein isoforms (p53, p53β, p53γ, 133p53, 133p53β, 133p53γ, 40p53, 40p53β and 40p53γ) due to alternative splicing and usage of the alternative promoter in intron 4. The expression of isoforms is tissue-specific, and their biological function is not fully understood. p53 gene has two relatives of major structural and functional similarities, p63 and p73. They also have a number of isoforms, which make p53 activity more complex. Luciferase assay was used to detect transcriptional activity of isoforms, p73β, p53, p53β, p53γ, 133p53, 133p53β, 133p53γ and 40p53 that were trasfected into H1299 cells, which lack expression of wildtype p53 protein. Transcriptional activity of each isoform was detected. p73β and p53β isoforms show the greatest degree of ranscriptional activity, compared to full-length p53. p53γ and 40p53 show a significant level of transcriptional activity, whereas 133p53, 133p53β and 133p53γ have extremely low levels of transcription activity. Western blot method has shown the expression of all isoforms at the protein level analysis.