神經膠質瘤之基因研究和治療:腫瘤抑制基因p16、p21、p53 及 Annexin7 之角色

Gene Study and Therapy for Glioma - Roles of Tumor Suppressor Genes p16, p21, p53, and Annexin7

洪國盛(Kuo-Sheng Hung)

指導教授 ： 洪純隆

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

無法控制之細胞增生是人類惡性腦瘤的特性。 腦瘤細胞生長極為快速，一部分是由於細胞內基因的改變，使得癌細胞不受正常之細胞循環所控制。 拜分子生物學進步之賜，使醫學界發現在癌細胞中，由於許多重要基因之改變，使得其細胞循環無法控制。 細胞循環是由一連串的蛋白脢(protein kinase)所調控，而此蛋白脢之活性乃是由周期素(cyclins)所控制。 Cyclins和周期素依賴酵素(cyclin-dependent kinases，CDKs) 協同作用於主要受質之磷酸化，使得細胞循環依序進行。　腫瘤抑制蛋白-Rb(retinoblastoma)是cyclin-CDK酵素群之重要標的，Rb蛋白之磷酸化使其對轉錄因子(transcription factor)(E2F家族)之抑制作用降低，造成細胞增生的基因表現增強。 除了cyclins和CDKs外，有一群CDK抑制因子(CDKIs)可調控cyclins和CDKs之活性。 顧名思義，CDK抑制因子可抑制cyclin-CDK複合體並傳達內在或外來之生長抑制訊息，此訊息可作用於細胞循環機制中。 因此，所有CDK抑制因子都可能成為腫瘤抑制基因。
吾人越來越明瞭癌細胞共同特性是細胞循環檢查點(check points)之失調，可能是由於正面調節因子(如，cyclins和CDKs)之異常表現;或者是負面調節因子之流失，包括藉由失去轉錄活性因子p53功能而鈍化的p21，及p16和Rb(retinoblastoma)等腫瘤抑制基因。
本章將詳細介紹正常細胞循環之知識和癌細胞中細胞循環如何受到干擾。 最近有許多報告指出在人類腦瘤中呈現細胞循環基因之表現的改變，所以，吾人會整理出腦瘤中正面和負面細胞循環調節因子之異常變化。 對於細胞循環內分子生物學之全盤了解使吾人發展出全新的基因治療來控制膠質神經質之增殖。
腫瘤抑制基因的突變或缺失在神經膠質瘤的生成和惡性化擔任了主要的角色。本文將比較以三種不同之腫瘤抑制基因(p53、 p21及p16)作用於大白鼠GBM細胞後，所產生抑制腫瘤細胞之功效及功能上表現之差異性。當腫瘤被帶有上述三種基因中之任何一種的人類全長之cDNA病毒感染後，發現這三種腫瘤抑制基因皆可造成腫瘤生成速率及腫瘤生成情形顯著降低。但是，此三種基因之腫瘤抑制效果有所不同：以p53為例，發現降低35%之腫瘤成長率、降低50%聚落形成率及減少了62%之腫瘤體積。至於p21基因成效則超越p53基因，p21基因之表現降低了91%之聚落形成率、降低66%之腫瘤成長率及降低了67%的腫瘤體積。至於p16之腫瘤抑制作用最為強烈，該基因表現可降低98%聚落形成率、減少60%腫瘤成長率減少90%之腫瘤體積。 p16和p21在細胞循環的調控路徑中, 屬下游階段，因此有較佳之腫瘤抑制作用，進而他們在各個抑制效果的機制亦有不同。本文證明使用腫瘤抑制基因p53、p21及p16可減少神經膠質瘤在活體外及活體內之成長。
腫瘤抑制基因在治療人類膠質母細胞瘤(GBM)上仍是重要而創新之利器。在很多人纇腫瘤中包括神經膠質瘤，黑色細胞瘤及白血病中可發現p16此腫瘤抑制基因之突變或缺陷。吾人利用大白鼠RT-1之GBM細胞株來研究p16基因是否可抑制GBM之生長。研究發現利用5 m.o.i.帶有人類之p16cDNA的反轉錄病毒(retrovirus)可以成功感染近100%之腫瘤細胞。這些感染的細胞中與對照組比較發現:感染病毒之腫瘤細胞在細胞聚落分析(colony forming assay)上有98%之抑制作用，另一方面在生長曲線(growth curve)上也有60%之抑制效果。利用西方點墨分析(Western blot analysis)發現外來性的p16蛋白之表現引致Rb蛋白之低磷酸化(hypophosphorylation)。與對照組比較，顱內注射p16感染之腫瘤細胞可造成大白鼠腫瘤體積95%之減少。在腫瘤中注射p16之retrovirus造成腫瘤細胞壞死, 也造成明顯人類p16基因之表現。在表現p16之RT-2腫瘤細胞中之無法偵測到細胞增生之標記PCNA，因此推論這些腫瘤細胞在p16基因表現後無法進入S phase。此外，多次之顱內注射p16 retrovirus可延長大白鼠之存活期達3.2倍。
在p16 retrovirus注射60 天後，10隻大白鼠中有2隻可發現停止生長之腫瘤。以上發現證明p16可有效抑制GBM之生長，其機制為p16表現使細胞停止G1 phase進而造成腫瘤細胞之生長降低以及壞死。本文證明使用腫瘤抑制基因p16可減少神經膠質瘤在活體外及活體內之成長。
最後吾人研究腫瘤抑制基因Annexin 7 (ANX7) 與人類膠質母細胞之預後關係。ANX7基因位於人類10q21之染色體上，此段染色體長久以來就被認定包含著與前列腺及其他腫瘤有關之腫瘤抑制基因。為了測試ANX7之表現是否與膠質母細胞瘤之預後有關，我們在一系列共99位病理檢體中，檢查ANX7, p53以及MIB-1之表現。在這些99位病人，我們以單變項以及多變項之分析來研究病人年齡，術前之綜合表現分數(KPS)，手術之範圍，腫瘤位置及免疫組織染色之特徵如ANX7，p53及MIB-1與存活期之關連性。Kaplan-Meier分析後顯示術前較高之KPS分數(P <0.0001)，全部腫瘤切除(P = 0.0072)，年輕人(P = 0.03)以及ANX7之表現乃是決定預後最重要之因子(P < 0.0001)。ANX7之表現與MIB-1免疫染色成正相關，但與p53無關。但是p53卻與MIB-1之染色呈正相關。這些發現顯示在膠質母細胞瘤之病人中術前較高之KPS，全部腫瘤切除，年輕族群及ANX7之表現，有較長之存活期。多變項之Cox回歸分析則指出在所有之變數中，ANX7之表現乃是與預後最有關之因子(P < 0.0001)。

關鍵字

腫瘤抑制基因 ； 基因治療 ； 神經膠質瘤

並列摘要

Uncontrolled cellular proliferation is the hallmark of human malignant brain tumors. Their growth proceeds inexorably, in part because their cellular constituents have an altered genetic code that enables them to evade the checks and balances of the normal cell cycle. Recently, a number of major advances in molecular biology have led to the identification of several critical genetic and enzymatic pathways that are disturbed in cancer cells resulting in uncontrolled cell cycling. We now know that the progression of a cell through the cell cycle is controlled in part by a series of protein kinases, the activity of which is regulated by a group of proteins called cyclins. Cyclins act in concert with the cyclin-dependent kinases (CDKs) to phosphorylate key substrates that facilitate the passage of the cell through each phase of the cell cycle. A critical target of cyclin-CDK enzymes is the retinoblastoma tumor suppressor protein, and phosphorylation of this protein inhibits its ability to restrain activity of a family of transcription factors (E2F family), which induce expression of genes important for cell proliferation. In addition to the cyclins and CDKs, there is an emerging family of CDK inhibitors, which modulate the activity of cyclins and CDKs. CDK inhibitors inhibit cyclin•CDK complexes and transduce internal or external growth-suppressive signals, which act on the cell cycle machinery. Accordingly, all CDK inhibitors are candidate tumor suppressor genes. It is becoming clear that a common feature of cancer cells is the abrogation of cell cycle checkpoints, either by aberrant expression of positive regulators (for example, cyclins and CDKs) or the loss of negative regulators, including p21Cip1 through loss of function of its transcriptional activator p53, or deletion or mutation of p16INK4A (multiple tumor suppressor 1/CDKN2) and the retinoblastoma tumor suppressor protein.
The mutation and/or deletion of tumor suppressor genes has been postulated to play a major role in the genesis and the progression of gliomas. In this study, the functional expression and efficacy in tumor suppression of 3 different tumor suppressor genes (p53, p21, and p16) were tested on a rat GBM cell line (RT-2). After the cells had been infected by a non-replicating virus with a full-length human cDNA insert of either p53, p21, or p16, significant reductions in both growth rate and tumor formation were found in all three genes examined. However, a difference in terms of suppressing power was observed. In the case of p53, a 35% reduction in growth rate, a 50% reduction in colony formation, and a reduction of 62% in tumor volume was seen. p21 superseded these effects with a 91% reduction of colony formation, 66% reduction of growth rate, and a 67% reduction in tumor volume. The most powerful suppressor effect is observed in the case of p16 with a 98% suppression in colony formation, a 60% reduction in growth rate, and a 90% reduction in tumor volume. This is not surprising, as p16 and p21 are more downstream in the cell cycle regulatory pathway as compared to p53. Moreover, the mechanism involved in each of their suppressor effects is different. This study demonstrates the feasibility of using tumor suppressor genes in regulating the growth of glioma in vitro and in vivo.

.Tumor suppressor genes may represent an important new therapeutic modality in the treatment of human glioblastoma (GBM). p16 INK4A is a tumor suppressor gene with mutation and/or deletion found in many human tumors, including glioblastomas, melanoma, and leukemias. RT-2 rat GBM cell line was used to investigate if p16 gene induces dominant suppression of glioblastoma growth. Close to 100% of tumor cells were infected by high titer pCL retrovirus encoding the full-length human p16 cDNA at 5 M.O.I.. Infected cells showed a 98% reduction in colony forming assay and a 60% reduction in growth curves in vitro compared to vector control. Exogenous overexpression of p16 induced hypophosphorylation of Rb protein by western blot analysis. Intracranial injection of p16 infected cells into syngeneic rats resulted in a 95% reduction in tumor volume compared to controls. Intratumoral injection of p16 retrovirus resulted in tumor necrosis and prominent human p16 transgene expressions. Proliferation marker PCNA was not detected in these human p16 expressed RT-2 tumor cells suggesting the cells were unable to enter into S phase after p16 expression. In addition, direct repeat intracranial injections of p16 retrovirus prolonged animal survival 3.2 folds compared to controls (48.4 ± 13.4 days vs 15.0 ± 2.1 days, p < 0.001). Two out of ten rats were found with dormant tumors at day 60 after p16 retrovirus injection. These results showed that p16 is effective in inhibiting GBM growth in situ. The mechanisms of tumor growth reduction and necrosis in vivo might be due to G1 arrest triggered by p16 expression.
Finally, we test a candidate tumor suppressor gene annexin 7 (ANX7) in 99 patinets with GBM, because GBM is the most common and lethal primary brain tumor in adults. It is nearly uniformly fatal, with a median survival of approximately one year, despite modern treatment modalities. However, a range of survival times exists around this median. Efforts to understand why some patients live longer or shorter than the average may provide insights into the biology of these neoplasms. The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor tumor suppressor genes associated with prostate and other cancers. To test whether ANX7 expression might be a predictor for glioblastomas, we examined the ANX7 expression, p53 accumulation, and MIB-1 labeling index in a retrospective series of 99 glioblastomas.
In all of the 99 cases, age, Karnofsky performance scale (KPS) index before surgery, extent of surgery, tumor location, and immunohistochemical features were analysed using univariate and multivariate analysis to identify whether any significance exists between ANX7 expression, p53 accumulation, MIB-1 labeling index and survival time. Kaplan-Meier analyses demonstrated that higher KPS before surgery (<0.0001), total tumor excision (p=0.0072), young age group (p=0.03), and ANX7 expression (p=0.0006) correlated with longer survival. Multivariate Cox regression analyses demonstrated that ANX7 expression was the strongest predictor of outcome (p<0.0001), independent of all other variables. In addition, ANX7 expression correlated with higher MIB-1 immunostaining, but did not correlate with p53 accumulation. Moreover, a significant positive correlation was observed between p53 and MIB-1 staining. These findings indicated that higher KPS before surgery, total tumor excision, young age group, and ANX7 expression correlated with longer survival in patients with glioblastomas. Multivariate Cox regression analyses demonstrated that ANX7 expression was the strongest predictor of outcome (p<0.0001), independent of all other variables.

並列關鍵字

p16 ； annexin7 ； gene therapy ； Glioblastoma ； tumor suppressor gene

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