Rad21與其裂解產物在乳腺上皮細胞之角色研究

Abstract

為了解Rad21在正常小鼠乳腺發育表現情形，與其N端與C端裂解產物是否會表現於不同發育階段乳腺中，本研究製備了具專一辨認N端或C端Rad21蛋白質之多株抗體。經由點墨吸漬法，證實了所製備出的抗N端與C端抗體與其抗原做結合能力皆具高感受性，抗體力價皆可達辨認1 pg抗原的能力。亦以免疫細胞螢光染色法證實了產製出的抗N端與C端抗體皆能辨認外源性Rad21與其N端或C端的重組蛋白。以西方吸漬法也證實能辨認到小鼠乳腺組織內源性的Rad21裂解產物。以西方吸漬法檢測基因表現量時發現，全長的Rad21大量表現於懷孕及泌乳期乳腺中，當乳腺開始進入離乳期時，全長Rad21表現量逐漸降低；然而，Rad21的N端與C端裂解產物表現量則在泌乳末期至離乳初期達到高峯，而後才隨離乳時間增加表現量逐漸下降。此一表現量變化，可能暗示著Rad21在乳腺發育過程扮演不同角色。為進一步的確認全長Rad21與其兩種裂解產物對乳腺上皮細胞株形態上的影響，當分別大量表現全長Rad21、N端與C端Rad21重組蛋白於人類乳腺上皮細胞株MCF-7中，以螢光免疫染色發現全長的Rad21蛋白大多座落於細胞核中，N端與C端裂解產物則分布於細胞核與細胞質中。除此之外，當以轉染方式表現C端Rad21蛋白於MCF-7細胞株中時，經由辨認活化細胞凋亡的執行蛋白caspase-3的抗體作螢光免疫染色，可偵測到表現C端Rad21的細胞亦同時表現活化的caspase-3蛋白，這顯示C端Rad21蛋白能誘導MCF-7進行計畫性細胞凋亡。為確認Rad21被蛋白酶截切與caspase-3間在發育乳腺中的相關性，以誘導細胞凋亡的藥物etoposide處理不朽化的乳腺上皮細胞株MCF-10A，以活化caspase-3的表現。經由西方吸漬法證實當MCF-10A被誘導發生細胞凋亡時，外源性表現的全長的Rad21可被截切產生一相似於C端Rad21裂解產物大小的片段。 綜合以上結果，全長Rad21在泌乳末期與離乳初期會被截切成N端與C端蛋白質產物，並從細胞核轉移至細胞質中。而C端Rad21蛋白即可能去誘導乳腺進行細胞凋亡，維持在不同發育階段乳腺細胞數量的穩定性。

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.