| Summary: | 碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 94 === Rad21 is one of the major cohesion subunits that holds chromatids together and controls separation of sister chromatids during mitosis. It is considered a novel nuclear caspase target molecule and plays a role in repairing DNA when it is damaged. Rad21 is cleaved by caspase-3 to promote apoptosis when breast cancer tumor suppressor protein (BRCA1) is phosphorylated in response to cell cycle and DNA damage in vivo. Unlike most other organs, development of the mammary gland occurs predominantly after birth. Once the gland is established, cycles of proliferation, functional differentiation, and apoptosis of alveolar epithelium occur repeatedly with each pregnancy. Thus, the regulation of apoptosis and cell proliferation closely affect alveolar epithelium development into normal acini or transforming into tumors during acinar formation. However, the function of Rad21 is not studied in detail in terms of mammary gland development. This study was undertaken to investigate the role of Rad21 and its cleavage products in mammary epithelial cells.
To begin with, we examined the expression pattern of Rad21 in different developmental stages of mammary gland to establish its correlative function in mammary glands. Two rabbit polyclonal peptide antibodies were developed that specifically recognizes the N-terminal or C-terminal of Rad21. To examine the specificity of these antibodies, full length, N and C-terminal Rad21 were transiently expressed in NIH3T3. The antibodies specifically recognized recombinant full length, N and C-terminal Rad21 proteins, as was detected using immuno-fluorescent staining techniques. The Rad21 expression profile was demonstrate with western blotting technique that was use to analyze Rad21 and its cleavage products’ protein expression profile in different stages of mice mammary gland development. The results showed that the full length Rad21 was detected in each developmental stage of mice mammary gland, however, it decreased from lactation day 14 to weaning day 4. On the other hand, cleaved N and C-terminal Rad21 increased from lactation day 14 to weaning day 2. These data indicate that Rad21 may have a special role to play in mammary gland development. To investigate the localization of Rad21 and its cleavage products in mammary epithelial cells, we transiently transfected full length N and C-terminal Rad21 into human mammary epithelial cell line MCF-7 and observed under immuno-fluorescent staining. The result suggested that the full length Rad21 was express only in the nucleus while N and C-terminal Rad21 were also present in the cytoplasm.Since the N and C-terminal Rad21 is highly expressed in late lactation and early weaning, we assume that they are involved in mammary gland involution. To address that, we transiently expressed Rad21 in MCF-7 cells and interestingly the data showed that only C-terminal Rad21 results in the induction of MCF-7 apoptosis as evidenced by the detection of active caspase-3 antibody with immuno-fluorescent staining. Furthermore, Rad21 was cleaved after inducing apoptosis by etoposide in immortalized human mammary epithelial cell line MCF-10A.
Combined together, these results show that, full length Rad21 is cleaved to generate N and C-terminal Rad21 that again are translocated from nucleus to cytoplasm, with the C-terminal Rad21 involved in promoting apoptosis of mammary epithelial cells. These data hint that the expression of C-terminal Rad21 in weaning of mammary gland may have a role in the regulation of mammary gland development.
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