DRUG RESISTANCE DUE TO LOSS OF BETA2 MICROGLOBULIN

癌症化疗的一个主要问题是固有的或获得性的耐药性。我们最近首次发现，β2微球蛋白(Beta2M)的缺失或表达降低与耐药的发生有关。这是一种全新的耐药机制，以前的文献中没有描述过。因此，该项目的总体目标是(1)揭开Beta2M导致耐药性的分子和生化机制，以及(2)开发由于Beta2M表达减少而导致的耐药性规避策略。其具体目的是：(1)确定细胞内β-2m的表达水平是否影响细胞周期；(2)确定β-2m在药物敏感细胞的凋亡中是否起作用，以及其表达的缺失或降低是否能阻止细胞的死亡；(3)通过细胞因子调节β-2m的表达，从而避免耐药；(4)研究耐药细胞中β-2m的表达调控，并确定这些细胞中β-2m基因抑制的分子机制。为了实现特定的目标1，将分别进行以下实验：(2)β2M是否分别调节特定细胞周期中特定细胞的功能，以及(B)β2M是否调节特定细胞周期控制蛋白的功能。为了达到特定的目的2，我们将通过(A)从正向或反义方向评估导入β2m基因的细胞中的水平，以及(B)检测在没有或存在化疗药物的情况下，抗β2m单抗是否诱导药物敏感的细胞而不是缺乏β2m的细胞，来探讨β2m在细胞凋亡中的作用。为了达到特定的目的3，我们将进行实验以评估干扰素-γ和肿瘤坏死因子-α在加或不加多柔比星或长春新碱的情况下对β2m表达降低的细胞和β2m转染体中的β2m表达的调节作用。在特定的目标4中，我们将确定(A)干扰素-γ或肿瘤坏死因子-α是否诱导特定的转录因子增强耐药细胞中β-2m基因的转录，以及(B)确定耐药细胞中β-2m的表达减少是由于正调控转录因子的减少或缺失，还是转录抑制因子的存在。这些研究将有助于了解这种新的耐药表型(S)由于β2m基因表达缺失或降低而产生的分子机制，并将有助于开发更有效的化疗或潜在的基因治疗策略。

A major problem in cancer chemotherapy is intrinsic or acquired drug resistance. We recently found, for the first time, that the loss or decreased expression of beta2- miocroglobulin (beta2M) is involved in the development of drug resistance. This is a completely novel drug resistance mechanism which has not been previously described in the literature. Therefore, the overall goals of this project are (1) to unravel the molecular and biochemical mechanisms by which beta2M causes drug resistance, and (2) to develop strategies to circumvent drug resistance due to the loss of decreased expression of beta2m. The Specific Aims are to (1) determine whether cellular levels of beta2m influence the cell cycle; (2) determine whether beta2m plays a role in apoptosis in drug sensitive cells, and whether its loss or decreased expression prevents apoptotic cell death; (3) modulate the expression of beta2m by cytokines and thereby circumvent drug resistance; and (4) investigate the regulation of beta2m expression in drug resistant cells, and determine the molecular mechanisms of beta2m gene suppression in these cells. In order to accomplish Specific Aim 1, experiments will be conducted to and decreases or increases cell proliferation, respectively, and (2) whether beta2m modulates the function of specific cell of specific cell-cycle, and decreases or increases cell proliferation, respectively, and (b) whether beta2m modulates the function of specific cell cycle-controlling proteins. To pursue Specific Aim 2, the role of beta2m in apoptosis will be explored by (a) evaluating levels in cells transfected with the beta2m gene in the sense or antisense orientation, and (b) examine whether anti-beta2m monoclonal antibodies in the absence or presence of chemotherapeutic agents, induce apoptosis in drug sensitive, but not in beta2m-deficient, cells. In order to accomplish Specific Aim 3, experiments will be conduced to evaluate the modulating effects of interferon-gamma (IFN- gamma) and tumor necrosis factor alpha (TNF-alpha), with or without doxorubican or vincristine, on beta2m expression in cells with reduced beta2m expression and in beta2m transfectants. In Specific Aim 4, we will determine whether (a) IFN-gamma or TNF-alpha induces specific transcription factors to enhance transcription of the beta2m gene in drug resistant cells, and (b) determine whether reduced expression of beta2m in resistant cells is due to decreased or absent positive regulatory transcription factors, or the presence of transcription suppressors. These studies will aid in understanding the molecular mechanism(s) of this novel drug resistance phenotype due to the loss or decreased expression of beta2m, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.