Objective The involvement of the 18-kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, in apoptosis regulation of HT29 colorectal cancer cells was studied in-vitro. In-vivo TSPO involvement in tumor growth of HT29 cells xenografted into SCID mice was studied. Methods Knockdown of TSPO expression in the human HT29 cell line was established by stable transfection with vectors containing the TSPO gene in the antisense direction. Successful TSPO knockdown was characterized by reduction of 20% in TSPO RNA levels, 50% in protein expression of the TSPO, and 50% in binding with the TSPO ligand, [3H]PK 11195. Subsequently, in-vitro cell viability and proliferation assays were applied. In addition, transient transfecton with short interfering RNA (siRNA) directed against human TSPO was studied in this way. Furthermore, we also grafted HT29 cells subcutaneously into the right thighs of SCID mice to examine the effects of the putative TSPO agonist, FGIN-1-27, on tumor growth in-vivo. Results In-vitro TSPO knockdown established by stable transfection of TSPO antisense gene resulted in HT29 clones displaying significantly lower levels of cell death as determined with trypan blue (50% less), lower apoptotic rates (28% less), and higher proliferation rates (48% more one week after seeding and 27% more two weeks after seeding). Transient transfection with anti-human TSPO siRNA resulted in similar viability and antiapoptotic effects. In-vivo, the proapoptotic TSPO ligand, FGIN-1-27 significantly reduced the growth rate of grafted tumors (40% less), in comparison with vehicle-treated mice. Conclusion TSPO knockdown by genetic manipulation transforms the human HT29 cancer line to a more malignant type in-vitro. In-vivo pharmacological treatment with the putative TSPO agonist FGIN-1-27 reduces tumor growth of the HT29 cell line. These data suggest that TSPO involvement in apoptosis provides a target for anticancer treatment.

The effect of unilateral eyestalk ablation on reproduction, molting and quality of offspring was investigated in Cherax quadricarinatus females during the reproductive-arrest period (winter). At the end of the Autumn, females that had spawned for the first time had relatively uniform, early vitellogenic ovaries, while most females that had spawned previously had ovaries containing two distinctly sized and coloured oocyte populations. These included one population of small oocytes, similar to oocytes found in first-time spawners, while the other comprised of large oocytes. Winter reproduction of females that had spawned previously was not affected by eyestalk ablation, whereas significantly increased spawning activity was observed in eyestalk-ablated first-time spawners. Both females that had spawned previously and first-time spawners that were eyestalk ablated molted significantly more frequently than intact females. The size and number of offspring were not affected by eyestalk ablation. These results show that small synchronous oocyte populations were affected by eyestalk ablation while large oocytes were not affected; females with single-sized-oocyte ovaries tended to be more receptive to the treatment. Unilateral eyestalk ablation may present a solution for induction of spawning only in young females and perhaps in synchronized broodstock.

Concurrent morphological, anatomical and physiological changes took place during the first reproductive cycle in the Australian red-claw crayfish Cherax quadricarinatus, which prepared the female for spawning and holding of the newly deposited eggs. The endopod became longer and wider than the exopod and developed a mixture of plumose and long thin simple (ovigerous) setae. Small oocytes (0.24 +/- 0.05 mm) were present in the immature ovary. The growing ovary contained two distinct oocyte populations: one consisted of small (0.55 +/- 0.07 mm), barely growing oocytes, while the other consisted of large oocytes, which increased in size continuously (0.73 to 2.55 mm) until egg laying took place. A gradual change in the relative abundance of ovarian polypeptides occurred until the late vitellogenic stage (large oocytes > 1.8 mm). Three predominant female-specific, SDS-PAGE separated, polypeptides were observed (103, 78 and 73 kDa) that may represent vitellin subunits. The most abundant carotenoid in the ovary was astaxanthin, while beta-carotene was present at a lower concentration. The strong correlation between the increasing diameter of the oocyte and the concentration of astaxanthin in the ovary and in the hemolymph suggested an association of astaxanthin with vitellin and vitellogenin.