33 research outputs found
Immortalized cells and one oncogene in malignant transformation: old insights on new explanation
<p>Abstract</p> <p>Background</p> <p>Nearly thirty years ago, it was first shown that malignant transformation with single oncogene necessarily requires the immortal state of the cell. From that time this thesis for the cells of human origin was not disproved. The basic point which we want to focus on by this short communication is the correct interpretation of the results obtained on the widely used human embryonic kidney 293 (HEK293) cells.</p> <p>Results</p> <p>Intensive literature analysis revealed an increasing number of recent studies discovering new oncogenes with non-overlapping functions. Since the 1970s, dozens of oncogenes have been identified in human cancer. Cultured cell lines are often used as model systems in these experiments. In some investigations the results obtained on such cells are interpreted by the authors as a malignant transformation of normal animal or even normal human cells (as for example with HEK293 cells). However, when a cell line gains the ability to undergo continuous cell division, the cells are not normal any more, they are immortalized cells. Nevertheless, the authors consider these cells as normal human ones, what is basically incorrect. Moreover, it was early demonstrated that the widely used human embryonic kidney 293 (HEK293) cells have a relationship to neurons.</p> <p>Conclusions</p> <p>Thus, the experiments with established cell lines reinforce the notion that immortality is an essential requirement for malignant transformation that cooperates with other oncogenic changes to program the neoplastic state and substances under such investigation should be interpreted as factors which do not malignantly transform normal cells alone, but possess the ability to enhance the tumorigenic potential of already immortalized cells.</p
Characterization of new cell line stably expressing CHI3L1 oncogene
Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1). Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development
3′-Hydroxymethyl 2′-deoxynucleoside 5′-triphosphates are inhibitors highly specific for reverse transcriptase
AbstractdNTP(3′-OCH3), a 3′-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase α, rat liver DNA polymerase β and calf thymus terminal deoxyribonucleotidyl transferase
Identification of diagnostic serum protein profiles of glioblastoma patients
Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer’s exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer’s exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome
Aim. To determine 293 and U373 cell response and ERK1/2 activation profile after CHI3L1 or CHI3L2 treatment. Methods. Specific activation and localization of ERK1/2 kinases after CHI3L1 or CHI3L2 addition to unsupplemented cell medium were evaluated by Western blots, immunofluorescence and confocal microscopy. To
determine whether CHI3L1 or CHI3L2 can enhance mitogenesis, [3H]thymidine incorporation in cellular DNA
was measured. Results. The obtained results show that ERK1/2 phosphorylation was stimulated in both cell types (293 and U373 cells) following addition of CHI3L2 or CHI3L1 in dose- and time-dependent manner. Unexpectedly, in opposite to CHI3L1, the dose-dependent decreasing of measured mitogenesis parameters was observed in 293 and U373 cells. In both cell types the treatment with CHI3L2 gave more sustained than CHI3L1
MAPK-pathway activation with prolonged phospho-ERK1/2 nuclear accumulation in 293 cells. Conclusions. In
contrast to the activation of ERK1/2 phosphorylation by CHI3L1 cell treatment, that leads to a proliferative signal, the activation of these kinases by CHI3L2 addition inhibits cell mitogenesis and proliferation in serum starved 293 and U373 cells.
Keywords: chitinase 3-like 1 protein (CHI3L1), chitinase 3-like 2 protein (CHI3L2), MAP kinase, mitogenesis.Мета. Визначити клітинну відповідь та встановити профіль активації кіназ ERK1/2 після обробки клітин 293 та U373 білками
CHI3L1 чи CHI3L2. Методи. Специфічну активацію і локалізацію кіназ ERK1/2 після внесення білків CHI3L1 або CHI3L2 до клітинного середовища, що не містить сироватки, проаналізовано
Вестерн-блот аналізо та методами імунофлуоресценції і конфокальної мікроскопії. Для встановлення, чи зможе додавання білків
CHI3L1 або CHI3L2 посилювати мітогенез, застосовано аналіз
інкорпорації [3H]тимідину в клітинну ДНК. Результати. Отримані результати демонструють, що стимуляція фосфорилювання кіназ ERK1/2 після додавання CHI3L1 або CHI3L2 має дозо- та
часозалежний характер. Виявилося, що, на відміну від CHI3L1, дозозалежне пригнічення інкорпорації [3H]тимідину констатовано у 293 та U373 клітинах. В обох типах клітин обробка білком
CHI3L2 викликає тривалішу активацію каскаду МАРК, ніж обробка білком CHI3L1, з акумулюванням кіназ ERK1/2 у ядрі клітин
293. Висновки. На противагу активації фосфорилювання кіназ
ERK1/2 внаслідок обробки клітин білком CHI3L1, що спричиняє
проліферацію, стимуляція цих кіназ додаванням білка CHI3L2 пригнічує мітогенез у клітинах 293 та U373, що перебувають у стані
сироваткового голодування.
Ключові слова: хітиназа 3-подібний білок 1 (CHI3L1), хітиназа 3-подібний білок 2 (CHI3L2), MAP-кіназа, мітогенез.Цель. Определить клеточный ответ и профиль активации киназ
ERK1/2 после обработки клеток 293 и U373 белками CHI3L1 или
CHI3L2. Методы. Специфическую активацию и локализацию киназ ERK1/2 после внесения белков CHI3L1 или CHI3L2 в клеточную среду, где отсутствует сыворотка, проанализировано методами Вестерн-блот анализа, иммунофлуоресценции и конфокальной микроскопии. Для определения, будет ли добавление белков
CHI3L1 или CHI3L2 увеличивать митогенез, использован анализ
инкорпорации [3H]тимидина в клеточную ДНК. Результаты. Полученные результаты демонстрируют, что стимуляция фосфорилирования киназ ERK1/2 после добавления CHI3L1 либо CHI3L2
имеет зависящий от дозы и времени характер. Оказалось, что, в
отличие от CHI3L1, дозозависимое угнетение инкорпорации [3H]
тимидина констатировано в 293- и U373-клетках. В обоих типах
клеток обработка белком CHI3L2 вызывает более длительную
активацию каскада МАРК, нежели обработка белком CHI3L1, с
аккумуляцией киназ ERK1/2 в ядрах клеток 293. Выводы. В отличие от активации фосфорилирования киназ ERK1/2 после обработки клеток белком CHI3L1, приводящей к пролиферации, стимуляция этих киназ добавлением белка CHI3L2 угнетает митогенез в клетках 293 и U373, находящихся в состоянии сывороточного голодания.
Ключевые слова: хитиназа 3-подобный белок 1 (CHI3L1), хитиназа 3-подобный белок 2 (CHI3L2), MAP-киназа, митогенез
Characterization of genes with increased expression in human glioblastomas.
peer reviewedIn the present study, we have used the gene expression data available in the SAGE database in an attempt to identify glioblastoma molecular markers. Of 129 genes with more than 5-fold difference found by comparison of nine glioblastoma with five normal brain SAGE libraries, 44 increased their expression in glioblastomas. Most corresponding proteins were involved in angiogenesis, host-tumor immune interplay, multidrug resistance, extracellular matrix (ECM) formation, IGF-signalling, or MAP-kinase pathway. Among them, 16 genes had a high expression both in glioblastomas and in glioblastoma cell lines suggesting their expression in transformed cells. Other 28 genes had an increased expression only in glioblastomas, not in glioblastoma cell lines suggesting an expression possibly originated from host cells. Many of these genes are among the top transcripts in activated macrophages, and involved in immune response and angiogenesis. This altered pattern of gene expression in both host and tumor cells, can be viewed as a molecular marker in the analysis of malignant progression of astrocytic tumors, and as possible clues for the mechanism of disease. Moreover, several genes overexpressed in glioblastomas produce extracellular proteins, thereby providing possible therapeutic targets. Further characterization of these genes will thus allow them to be exploited in molecular classification of glial tumors, diagnosis, prognosis, and anticancer therapy
Origin of Insulin Receptor-Like Tyrosine Kinases in Marine Sponges
Volume: 197Start Page: 198End Page: 20