During cancer therapy with DNA-damaging drug-agents, the development of secondary resistance to apoptosis can be observed. In the search for novel therapeutic approaches that can be used in these cases, we monitored chemotherapyinduced apoptosis resistance in a syngenic mouse tumor model. For this, syngenic murine colorectal carcinoma cells, which stably expressed a FRET-based caspase-3 activity sensor, were introduced into animals to induce peritoneal carcinomatosis or disseminated hepatic metastases. This syngenic system allowed in vitro, in vivo and ex vivo analysis of chemotherapy induced apoptosis induction by optically monitoring the caspase-3 sensor state in the tumor cells. Tumor tissue analysis of 5-FU treated mice showed the selection of 5-FU-induced apoptosis resistant tumor cells. These and chemo-naive fluorescent tumor cells could be re-isolated from treated and untreated mice and propagated in cell culture. Re-exposure to 5-FU and second line treatment modalities in this ex-vivo setting showed that 5-FU induced apoptosis resistance could be alleviated by imatinib mesylate (Gleevec). We thus show that syngenic mouse systems that stably express a FRET-based caspase-3 sensor can be employed to analyse the therapeutic efficiency of apoptosis inducing chemotherapy.
Since the beginning prion research has been largely dependent on animal models for deciphering the disease, drug development or prion detection and quantification. Thereby, ethical as well as cost and labour-saving aspects call for alternatives in vitro. Cell models can replace or at least complement animal studies, but their number is still limited and the application usually restricted to certain strains and host species due to often strong transmission barriers. Bank voles promise to be an exception as they or materials prepared from them are uniquely susceptible to prions from various species in vivo, in vitro and in cell-free applications. Here we present a mainly astrocyte-based primary glia cell assay from bank vole, which is infectible with scrapie strains from bank vole, mouse and hamster. Stable propagation of bank vole-adapted RML, murine 22L and RML, and hamster 263K scrapie is detectable from 20 or 30 days post exposure onwards. Thereby, the infected bank vole glia cells show similar or even faster prion propagation than likewise infected glia cells of the corresponding murine or hamster hosts. We propose that our bank vole glia cell assay could be a versatile tool for studying and comparing multiple prion strains with different species backgrounds combined in one cell assay.
If genetic lesions were the sole reason of damage induced by ionizing radiation, an increase in the number of identical chromosome sets (polyploidy) may be expected to have a radioprotective effect. This effect is evident in terminally differentiated tissues when the reduction in remaining life span is used as the criterion. This effect is also evident in cells capable of proliferation if cytoplasmic growth during the period of mitotic delay is restricted and the criterion used is continuation of cell proliferation. Both instances demonstrate that polyploidy, in principle, can exert a radioprotective effect, although the genetic damage induced by a given dose increases in approximate proportion to ploidy. However, in mitotically active cells, without restrictions in cytoplasmic growth, differentiation enhancement dominates the effects of genetic lesions, and polyploidy does not protect. Enhancement of differentiation causes damage by eliminating amplification divisions normally passed through by cell progenies before terminal differentiation, thus reducing the number of differentiated cells produced. From its dependence on excess cytoplasmic growth it is concluded that the phenomenon is caused by the interference of ionizing radiation with a mechanism that provides intracellular signals needed to coordinate molecular interactions involved in the control of cell differentiation. This conclusion corresponds to experiments that suggest that intracellular control of differentiation depends on an increase in the ratio of essential cytoplasmic constituents, probably mitochondrial genomes, per nuclear genome. The action of chemical differentiation enhancing agents is similar and an outline of probable mechanisms is presented. Regarding late radiation damage it is concluded that non-specific genetic lesions can enhance differentiation by permanently prolonging the cell cycle, which causes an increased cytoplasmic growth rate per cycle. In this case polyploidy cannot protect because the induced genetic lesions are proportional to ploidy. Both the duration of mitotic delay, and the extent of genetic lesions increase with chromosome size, thus explaining the correlation between interphase chromosome volume and radio-sensitivity. Lack of substantial radioprotecting effect of polyploidy in neoplastically transformed mammalian cells indicates residual capabilities to cease cell proliferation by mechanisms related to terminal differentiation, thus offering clues to tumour therapy.
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