Podcasts about ht29

In der vorliegenden Arbeit wurde die Wirkung der Farnesyltransferase-Inhibitoren hinsichtlich der antitumorösen Wirkung auf gastrointestinale Tumore in verschiedenen in vitro Experimenten untersucht. Als Marker für die therapeutische Wirkung wurde die Induktion der Apoptose gewählt. Durchgeführt wurden unsere Versuche mit den Zelllinien HepG2, gewonnen aus einem hepatozellulären Karzinom, sowie HT29, einem kolorektalen Adenokarzinom, unter Verwendung von BMS-214662 und SCH66336 als Vertreter der Farnesyltransferase-Inhibitoren. Diese werden derzeit in klinischen Studien getestet. BMS-214662 befindet sich in Phase II, im Rahmen von SCH66336 sind bereits Phase III Studien abgeschlossen worden. Anhand von Apoptose-Assays waren wir in der Lage einen antitumorösen Effekt durch Induktion der Apoptose aufzuzeigen. Hierbei war die Apoptose-Auslösung dosisabhängig von dem verwendeten Farnesyltransferase-Inhibitor. Im Gegensatz zu SCH66336 induzierte BMS-214662 bereits unter Verwendung von 1µM in 24h die Apoptose, wohingegen eine Konzentration von 25µM SCH66336 notwendig war, eine signifikante Apoptose hervorzurufen. Die Induktion der Apoptose war dabei unabhängig von einer Ras-Mutation. Sowohl in den HT29-Zellen mit Mutation des Ras-Proteins, als auch in den mutationsfreien HepG2-Zellen erfolgte ein Anstieg der Apoptoserate. Zusätzlich zeigten wir einen möglichen synergistischen Effekt für den Antikörper anti-APO in Verbindung mit BMS-214662. Mit Hilfe des Western Blots konnte als ein möglicher Induktionsfaktor eine gesteigerte Rekrutierung der Caspase 8 dargestellt werden. Dies bestätigte sich durch eine messbare Erhöhung der Caspase 3, welche durch Caspase 8 aktiviert wird und den apoptotischen Tod der Zelle hervorruft. Insgesamt läuft der Mechanismus Rezeptor- bzw. Liganden-unabhängig ab, da in der durchgeführten rt-PCR weder vermehrt CD95-Rezeptor bzw. dessen Ligand nachgewiesen werden konnte.

Background: The shape of the dose-response curve at low doses differs from the linear quadratic model. The effect of a radio-adaptive response is the centre of many studies and well known inspite that the clinical applications are still rarely considered. Methods: We studied the effect of a low-dose pre-irradiation (0.03 Gy-0.1 Gy) alone or followed by a 2.0 Gy challenging dose 4 h later on the survival of the HT29 cell line (human colorectal cancer cells) and on the GM637 cell line (human fibroblasts). Results: 0.03 Gy given alone did not have a significant effect on both cell lines, the other low doses alone significantly reduced the cell survival. Applied 4 h before the 2.0 Gy fraction, 0.03 Gy led to a significant induced radioresistance in GM637 cells, but not in HT29 cells, and 0.05 Gy led to a significant hyperradiosensitivity in HT29 cells, but not in GM637 cells. Conclusion: A pre-irradiation with 0.03 Gy can protect normal fibroblasts, but not colorectal cancer cells, from damage induced by an irradiation of 2.0 Gy and the application of 0.05 Gy prior to the 2.0 Gy fraction can enhance the cell killing of colorectal cancer cells while not additionally damaging normal fibroblasts. If these findings prove to be true in vivo as well this may optimize the balance between local tumour control and injury to normal tissue in modern radiotherapy.

The human colon cancer cellline HT29 was investigated in various models regarding different criteria. The in-vivo-experiments were carried out with female SCID mice and consisted of subcutaneous cell injection, orthotopic cell injection into the cecal wall and orthotopic fixation of a tumor fragment onto the cecum. The subcutaneous experiment took 41 days; the orthotopic animal experiments were equally divided into three points of necropsy each two weeks apart. On these days tumor, liver and lung were withdrawn, fixed in 3,8% formaldehyde and analyzed histologically. In addition blood samples of all animals of the orthotopic experiments were taken on days of autopsy and the CEA (carcinoembryonic antigen) content was determined using an ELISA. The vascularization of the orthotopic primary tumors was examined by staining of CD34, i.e. number and area of the tagged vessels were ascertained. Additionally the green fluorescent protein (GFP) was studied in view of its suitability as quantifiable reporter gene in these models. Therefore not only HT29-wildtype but also HT29 cells transfected with GFP were used in vitro and in the first two in vivo assays. Advantages and disadvantages summarized: - The subcutaneous model was realized easily, measurement of the primary tumor was simple, the tumor take rate was 100 % and the laboratory animals appeared to suffer only from a relative slight amount of stress. Besides these advantages this setting cannot be used for investigations regarding metastasis because of the low number of metastases. - The orthotopic cell injection generated small, hardly measurable primary tumors, but this approach is a suitable model of metastasis because of the number of metastases detected. The technical effort and the burden for the animals exceeded that found in the subcutaneous setting but was below the effort of orthotopic fixation of a tumor fragment. - Of all investigated models the orthotopic fixation of a tumor fragment represents the model with the greatest effort. The primary tumors were big enough to be measured, but the number of metastases was too low to make statistical valuable evaluations. - The CD34 staining successfully marked the vessels of the primary tumor and facilitated a computer-assisted quantitative analysis of the vascularization of orthotopic colon tumors. It was assessed that a broad neoangiogenesis occurred at the beginning of tumor growth prior to the development of metastases. The number of metastases increased with proceeding length of time, whereas the number of vessels decreased. The continuous extension in tumor volume resulted in a necrotic tumor center so that vessels were detectable in the border area only. - The used HT29-GFP clone was not stable enough to generate sufficient fluorescence. The cells both in vitro an in vivo grew more slowly, the subcutaneous tumors showed necrotic areas and there were less metastases after orthotopic injection of GFP cells than after injection of wildtype cells. Because of the insufficient fluorescence it was not possible to execute a quantifiable analysis of metastasis. The application of GFP was not advantageous within these models. - CEA suits to be a valuable tumor marker for colon cancer in both investigated models. The CEA content in the blood samples of tumor bearing animals increased dependent on tumor burden and tumor invasiveness. A direct correlation between tumor size and CEA might be established with an improved measurement system or rather an advanced measuring of tumor volume. Due to the comparative characterization of injection and implantation techniques as well as other detailed examinations carried out for this thesis, it is possible to select suitable models for preclinical trials depending on the individual purpose.