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|Title:||A study of the relationship among all-trans retinoic acid, arginase I and apoptosis : implication of arginase I in controlling cell death||Authors:||Chow, Ho-yin||Keywords:||Hong Kong Polytechnic University -- Dissertations
Tretinoin -- Therapeutic use
Enzymes -- Therapeutic use.
Apoptosis -- Research.
Cells -- Growth -- Research
|Issue Date:||2010||Publisher:||The Hong Kong Polytechnic University||Abstract:||All-trans-retinoic acid (RA) is able to induce cell growth inhibition or apoptosis in many kinds of malignant cells such as hepatocarcinoma, melanoma and leukemia. Previously, our collaborators found that when RA was injected into pregnant mice, the embryos showed caudal regression due to apoptosis in the early stage of embryogenesis. Just right before apoptosis took place, we found by differential display that the arginase I gene was up-regulated 5 h after RA was injected. Therefore, we hypothesized that RA is able to induce arginase I up-regulation directly which consequently stimulates apoptosis in some cells. In this thesis, we aimed to find out whether arginase is involved in RA-induced cell death or cell growth inhibition in mouse embryonic carcinoma cells P19 and melanoma B16-F0 (B16) cells, and the possible occurrence of the retinoic acid response element (RARE) in the promoter region of the mouse arginase I gene. In addition, the role of arginase, either intra-cellularly induced or extra-cellularly added, would be discussed. Both P19 and B16 cells were exposed to different concentrations of RA for 24-72 h. Arginase mRNA expression was detected by real-time PCR and its enzyme activity by activity assay. Cell growth inhibition was measured by MTT viability assay. Apoptosis of cells was measured by Annexin V-FITC apoptosis staining kit using flow cytometry. After exposure to RA, both P19 and B16 cells showed significant up-regulation of arginase I mRNA expression, but not arginase II, in a time- and dose-dependent manner, and the elevated arginase activity induced by RA paralleled with cell growth inhibition of both cell lines. In P19 cells, arginase I mRNA was induced in 12 h but apoptosis was stimulated in 18 h. Surprisingly, RA stimulated time-dependent apoptosis in P19 cells but only induced time-dependent growth inhibition without apoptosis in B16 cells. To test the arginase dependency of RA-induced apoptosis in P19 cells and cell growth inhibition in B16 cells, arginase inhibitor was added together with RA and apoptosis and cell viability assay was performed. Apoptosis was reduced when Nω-hydroxy-nor-arginine (norNOHA) was added together with RA in P19 cells. Cell growth was restored when arginase inhibitor norNOHA or Nω-hydroxy-L-arginine (NOHA) was added in addition to RA in B16 cells. L-ornithine and polyamines, the products of arginase-ornithine decarboxylase (ODC) pathway, could also induce either apoptosis in P19 cells or cell growth inhibition in B16 cells. RA-induced apoptosis in P19 cells was abolished by inhibition of ODC using specific ODC inhibitor a-Difluoromethylornithine (DFMO) but was restored by polyamines, indicating the effectors in the relationship between RA, arginase and apoptosis or cell growth inhibition in the two cell lines.
The data of the reporter assay using luciferase system showed that there was a consensus RARE sequence located in the promoter region of mouse arginase I gene. In addition, arginase I mRNA was still up-regulated by RA in the presence of protein synthesis inhibitor cycloheximide (CHX), indicating that RA induces arginase I transcription directly and requires no de novo protein synthesis. Since arginase was up-regulated by RA and stimulated apoptosis in P19 cells, we tested whether arginase induced by other molecules would result in apoptosis as well. Cyclic AMP (cAMP), a well-known and patented arginase-inducing agent, was added to P19 cells for qRT-PCR assay, arginase activity assay and apoptosis assay. Consistent with other published data, arginase activity was up-regulated in P19 cells except that both arginase I and arginase II mRNA levels were found to account for the up-regulation of activity. To check if cAMP-induced apoptosis in P19 cells was arginase-dependent, cAMP was added to the cells in the presence of norNOHA. Apoptosis was abolished by the inhibition of arginase, similar to RA-induced apoptosis, indicating that once arginase was up-regulated in P19 cells, cells would undergo apoptosis. Besides intra-cellular arginase gene induction, arginase protein was added to the growth medium of P19 and B16 cells to check if there would be apoptosis and cell growth inhibition. After bovine arginase was added to the of P19 cells for 24 h, arginine in the culture medium was greatly reduced with the elevation of ornithine. Moreover, we observed cell cycle arrest in the S phase and the G₀/G₁ phase of P19 and B16 cells, respectively. Both cell lines showed apoptosis. Cell death was induced by depleting arginine in the growth medium because the cells were found to depend on the availability of arginine in the growth medium. For the first time, these data suggest that both intra-cellular and extra-cellular arginase stimulate apoptosis in P19 cells or growth inhibition in B16 cells. The expression modulation of arginase may provide insight into treatment of arginase-related diseases.
|Description:||xix, 201 p. : ill. (some col.) ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577P ABCT 2010 Chow
|URI:||http://hdl.handle.net/10397/3343||Rights:||All rights reserved.|
|Appears in Collections:||Thesis|
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