DRUG RESISTANCE DUE TO LOSS OF BETA2 MICROGLOBULIN

癌症化疗的一个主要问题是内在或获得性耐药性。我们最近首次发现，β 2-微球蛋白（β 2 M）表达的缺失或减少与耐药性的发展有关。这是一种全新的耐药机制，以前在文献中没有描述过。因此，本项目的总体目标是（1）阐明β 2 M导致耐药性的分子和生化机制，以及（2）开发策略以规避由于β 2 M表达减少而导致的耐药性。具体目的是（1）确定β 2 m的细胞水平是否影响细胞周期;（2）确定β 2 m是否在药物敏感细胞的凋亡中起作用，以及其表达缺失或降低是否阻止凋亡性细胞死亡;（3）通过细胞因子调节β 2 m的表达，从而避免耐药性;（4）研究β 2 m基因在耐药细胞中的表达调控，探讨β 2 m基因在耐药细胞中被抑制的分子机制。为了实现特定目的1，将进行实验以分别降低或增加细胞增殖，和（2）β 2 m是否调节特定细胞周期的特定细胞的功能，和分别降低或增加细胞增殖，和（B）β 2 m是否调节特定细胞周期控制蛋白的功能。为了追求特定目标2，将通过以下方式探索β 2 m在细胞凋亡中的作用：（a）评价用有义或反义方向的β 2 m基因转染的细胞中的水平，和（B）检查在不存在或存在化疗剂的情况下，抗β 2 m单克隆抗体是否诱导药物敏感性细胞的细胞凋亡，但不诱导β 2 m缺陷细胞的细胞凋亡。为了实现特定目标3，将进行实验以评价干扰素-γ（IFN-γ）和肿瘤坏死因子α（TNF-α）（有或没有多柔比星或长春新碱）对β 2 m表达降低的细胞和β 2 m转染子中β 2 m表达的调节作用。在具体目标4中，我们将确定（a）IFN-γ或TNF-α是否诱导特异性转录因子以增强耐药细胞中β 2 m基因的转录，以及（B）确定耐药细胞中β 2 m表达降低是否是由于正调控转录因子减少或缺失，或者转录抑制因子的存在。这些研究将有助于理解由于β 2 m表达缺失或降低而导致的这种新型耐药表型的分子机制，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.