Tumor molecular margins

Zmiany molekularne charakterystyczne dla guza nowotworowego, takie jak mutacje genetyczne czy zmieniony poziomswoistych transkryptów i białek, obserwowane są niekiedy w obrębie prawidłowych morfologicznie i histologicznietkanek otaczających nowotwór. Taki obszar tkanek otaczających nowotwór nazywany jest marginesem molekularnymguza. Zakłada się, że zmiany molekularne obserwowane w tym obszarze mogą być etapem poprzedzającymzmiany morfologiczne i histologiczne. Skuteczność operacyjnego leczenia nowotworów zależy od całkowitej resekcjiguza, a przyczyną niepowodzenia leczenia mogą być wznowy miejscowe, będące efektem pozostawienia pozapolem operacyjnym komórek nowotworowych. Nie można jednak wykluczyć, że wznowy miejscowe są efektemtransformacji nowotworowej sąsiadujących z guzem tkanek histologicznie prawidłowych, zainicjowanej występującymitu wcześniej zmianami molekularnymi. W takim ujęciu zdefiniowanie marginesów molekularnych guza i ichusunięcie razem z nowotworem może być kluczowe dla skuteczności leczenia operacyjnego. Istnieje szereg narzędzidiagnostycznych umożliwiających zdefiniowanie obszaru i określenie właściwości molekularnych marginesów guzanowotworowego. Do najczęściej stosowanych należą metody immunohistochemiczne i metody oparte na reakcjiPCR, umożliwiające analizę wybranych białek, transkryptów i genów. W ostatnim czasie podejmowane są równieżpróby wykorzystania do badania marginesów molekularnych guza nowotworowego zaawansowanych techniczniemetod genomiki i proteomiki. Do najbardziej obiecujących wśród nich należy metoda obrazowania molekularnegotkanek za pomocą spektrometrii mas (Imaging Mass Spectrometry).Molecular changes characteristic of malignant tumors such as genetic mutations or altered levels of specific transcriptsand proteins are sometimes observed within the morphologically and histologically normal tissue adjacent to thetumor. Such an area of morphologically normal yet molecularly changed tissue is called a molecular margin of tumor.It has been suggested that molecular changes observed in this area might be followed by a further transformationinto a malignant tumor. The efficacy of surgical treatment of the tumor depends on complete resection of cancercells, hence local recurrence and failure of the treatment might result from tumor cells residing outside the resectedarea. However, a local recurrence could also result from malignant transformation of cells in morphologically andhistologically normal tissue adjacent to the tumor where molecular cancer-initiating changes were pre-existing. Henceidentification and delineation of molecular margins of tumor (and removing them together with the cancer) mightbe crucial for the efficacy of the treatment. There are several diagnostic tools that can be used for identification andcharacterization of tumor molecular margins. The most commonly used include immunohistochemistry and PCR-basedmethods, which allows analysis of pre-selected proteins, transcripts and genes. Most recently, technically advancedmethods of genomics and proteomics have been also proposed in studies of molecular margins of cancer, includingmolecular imaging of tissue by mass spectrometry (so called Imaging Mass Spectrometry)

High mobility group proteins stimulate DNA cleavage by apoptotic endonuclease DFF40/CAD due to HMG-box interactions with DNA

The DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. It has been previously demonstrated that the major HMG-box-containing chromatin proteins HMGB1 and HMGB2 stimulate naked DNA cleavage by DFF40/CAD. Here we investigate the mechanism of this stimulation and show that HMGB1 neither binds to DFF40/CAD nor enhances its ability for stable binding to DNA. Comparison of the stimulatory activities of different truncated forms of HMGB1 protein indicates that a structural array of two HMG-boxes is required for such stimulation. HMG-boxes are known to confer specific local distortions of DNA structure upon binding. Interestingly, the presence of DNA strand cross-links formed by cisplatin or transplatin, which may somehow mimic distortions induced by HMG-boxes, also affects DNA cleavage by the nuclease. The data presented suggest that changes induced in DNA conformation upon HMG-box binding makes the substrate more accessible to cleavage by DFF40/CAD nuclease and thus may contribute to preferential linker DNA cleavage during apoptosis

Identification of novel putative regulators of the major apoptotic nuclease DNA Fragmentation Factor

Yeast two- and three-hybrid systems were used to screen cDNA libraries from HeLa cells and human brain tissue to identify novel protein partners of DNA Fragmentation Factor, the major apoptotic nuclease. The two-hybrid system revealed the DFF45 inhibitory subunit of the nuclease as the only identified partner of the DFF40 catalytic subunit. Similar analysis revealed several protein candidates that potentially interact with the DFF45 subunit: FBXO28, FOSL1, PGK1, PCNT, FHL1 and GFAP. Recombinant GFAP protected DFF45 against cleavage with caspase-3 and prevented activation of the DFF nuclease in vitro. In addition, three-hybrid system results revealed a putative novel protein partner of the DFF40-DFF45 heterodimer. The candidate cDNA contained two open reading frames that mapped to an intron of the GBF1 gene. Products of the candidate cDNA derived from a cell-free transcription/translation system inhibited DNA cleavage by recombinant caspase-activated DFF. This putative partner of DFF may have functional importance in regulating the apoptotic response because its RNAi silencing facilitated cleavage of the DFF45 inhibitor subunit and affected chromatin fragmentation in HeLa cells undergoing apoptosis. This hypothetical protein, named DRIG based on an acronym specifying its genomic location, could be a novel factor involved in regulation of DFF40 apoptotic nuclease

TNFα-induced activation of NFκB protects against UV-induced apoptosis specifically in p53-proficient cells

The signaling pathways that depend on p53 or NFκB transcription factors are essential components of cellular responses to stress. In general, p53 is involved in either activation of cell cycle arrest or induction of apoptosis, while NFκB exerts mostly anti-apoptotic functions; both regulatory pathways apparently interfere with each other. Here we aimed to analyze the effects of NFκB activation on DNA damage-induced apoptosis, either p53-dependent or p53-independent, in a set of human cell lines. Four cell lines, HCT116 and RKO colon carcinoma, NCI-H1299 lung carcinoma and HL60 myeloblastoma, each of them in two congenic variants either containing or lacking transcriptionally competent p53, were used. Cells were incubated with TNFα cytokine to activate NFκB and then treated with ultraviolet or ionizing radiation to induce apoptosis, which was assessed by measurement of the sub-G1 cell fraction. We observed that treatment with TNFα resulted in a significant reduction in the frequency of apoptotic cells in UV-irradiated p53-proficient lines (with exception of the UV-resistant NCI-H1299 cells). This anti-apoptotic effect was lost when cells were pretreated with parthenolide, an inhibitor of NFκB activation. In marked contrast, TNFα-pretreatment of p53-deficient lines resulted in an increased frequency of apoptotic cells after UV irradiation (with exception of HL60 cells). Such anti- and pro-apoptotic influence of TNFα was less obvious in cells treated with ionizing radiation. The data clearly indicates functional interference of both signaling pathways upon the damage-induced apoptotic response, yet the observed effects are both cell type- and stimulus-specific