細胞週期核蛋白CANP在癌症細胞之角色

Abstract

肝細胞癌 (HCC)是台灣最常見的惡性腫瘤。為了評估基因異常表現對於肝細胞癌影響，我們使用了mRNA基因分析法(Differential display)在肝細胞癌組織中辨認到一個異常表現，而且功能不明的新穎基因，它是位於染色體11q12.2。在我們過往的研究中，展示出它的基因產物會落在細胞核，並把它命名為Cancer Associated Nuclear Protein (CANP)。 在本次的研究裡，我們首先對比人類CANP蛋白與其他物種的親源性關係。在利用電腦軟體的蛋白序列分析中，可以找到CANP蛋白上在多個與細胞週期有關的磷酸化的位置。而且，CANP蛋白序列上也被發現帶有KEN-box和D-box，這發現提供了CANP蛋白也許是APC-cdh-1或APC-cdc20的受質，並經由ubiquintin-proteasome路經所分解的可能性。另一方面，我們也從CANP蛋白序列上找到四個可以被sumoylation位置，不過，以免疫沈澱(Immunoprecipitation,IP)及西方點墨法 (Western blot)進行的實驗裡，並無法證明CANP蛋白有被sumoylation。再者，我們證明了CANP擁有一個比較短，缺乏編碼順序三 (Exon 3)的選擇性剪裁變體 (Alternative splicing variant)。 利用HeLa細胞株的細胞週期進行，我們展示了CANP的mRNA和蛋白量會在G0/G1期開始上升，直到S期達到最高峰，然後開始下降到M/G1期的最低點。而CANP在細胞週期進行時，它會表現出一個獨特的分佈模式。當細胞處於細胞分裂時期，CANP會擴散至細胞質，並且形成一個環狀排列，包圍染色體，稍後就會集中至中央體 (Centrosome)。當細胞進入分裂期間 (Interphase)，CANP形成大夥粒狀，變得集中在核仁位置。 為了說明CANP蛋白在腫瘤細胞生長時的功能，我們利用RNAi oligos去調降 (Knockdown) CANP mRNA的表現量。結果發現CANP與細胞在半固體瓊脂培養基的非貼附性生長能力及利用MTT測量細胞增生速率的實驗中並無明顯功能。然而，在CANP被RNAi oligos調降的細胞株內 (HeLa, HA22T, HCC36, HBL435及Au565)顯現出極大的侵犯能力(Invasion capacity)下降。而且也發現有好幾種金屬蛋白分解脢 (Matrix metalloproteinase, MMP)的mRNA量在CANP被低度調控後，而有所下降。另外我們也發現在CANP mRNA的調降後,兩種與細膩移動有關的細胞週期蛋白：cyclin B2 及cdc2的蛋白量也有被抑制的現象。這個結果或許暗示了各細胞移動能力和株侵犯能力的下降是與金屬蛋白分解脢的mRNA量和cyclin B2及cdc20的蛋白量下降有關。 數種參與細胞週期有關的重要蛋白，例如週期素 (Cyclin A1, Cyclin B1)，在細胞的CANP被低度調控之後都有累積的情況。這表示CANP也許有細胞週期中扮演著某個角色。另外，我們觀察當細胞曝露於紫外線之後三十分鐘，CANP的mRNA量有被減少的情形，但是在兩個小時以後，CANP的mRNA量又會有反彈的現象，這個結果意謂著CANP對於紫外線引致的DNA損壞有反應，並且可能參與損壞DNA的修補。

Hepatocellular carcinoma (HCC) is the most common fatal malignancy in Taiwan. To elucidate the aberrant genes expression in HCC, we used mRNA differential display and identified a novel gene with unknown functions which was frequently over-expressed in HCC, which is located at chromosome 11q12.2. Our previous study showed that the gene product was located to the nuclei, and named as Cancer Associated Nuclear Protein (CANP). In this study, we first compared the human CANP protein with homologues of other species. The peptide sequence analysis predicted several putative phosphorylation sites, including some are cell cycle related. Moreover, CANP protein contains KEN box and D-box motifs which suggests that CANP maybe a novel substrate of APC-cdh-1 or/and APC-cdc20 and degradated through the ubiquitin-proteasome pathway. There were also four possible sumoylation sites, which, however, could not be verified by immunoprecipitation (IP) and western blot using sumoylation assay. Besides, we showed that CANP had a shorter alternative splicing variant, which lacked the exon-3. Using the standard cell cycle progression of HeLa cells, we showed the mRNA level and protein levels of CANP increased dramatically from the G1/S phase transition and reached the peak in the S phase, and then decreased to the lowest level when cells in the M/G1 phase. Further, CANP exhibited a unique distribution pattern throughout the cell cycle, during the mitosis, CANP protein became dispersed in the cytoplasm, with weak ring-shape concentration around the chromatin, and marked concentration in the centrosomes. While in the interphase nuclei, CANP became condensed as large granules corresponding to the nucleoli. To elucidate the function(s) of CANP on the tumor cell growth, we used RNAi oligos to knockdown the expression of CANP. There was no significant difference on the anchorage independent growth in soft agar assay and cell proliferation on MTT assay. However, the CANP RNAi oligos knockdown cells (HeLa, HA22T, HCC36, HBL435 and Au565) exhibited greatly reduced invasion capacity on the in vitro invasion assay. And there were several Mmps which were down-regulated after the CANP silencing. Moreover, we found that the protein level of two cell cycle related protein: cyclin B2 and cdc20 was depressed which were related to the cell migration after the CANP knockdown. It suggested that the reduction of cell migration and invasion capacity maybe caused by the depression of Mmps and the protein level of cyclin B2 and cdc20. Several important cell cycle related proteins, including cyclin A1 and cyclin B1. They accumulated after the suppression of CANP. These findings suggest that CANP plays a role in cell cycle. Notably, we also observed that the mRNA level of CANP was suppressed in 30 minutes after the UV exposure, but became rebound two hours later. These results implicate that CANP is sensitive to UV triggered DNA damage and probably involved in the damage repair.