AKT PROTOONCOGENE IN BREAST CANCER

这项建议将解决这样的假设，即AKT1，即蛋白-丝氨酸/苏氨酸激酶癌基因v-AKT的细胞同源基因，在乳腺癌的增殖、生存和病因学中发挥关键作用。这将通过以下具体目标来解决：1)确定AKT1，以及可能的其他AKT亚型，在依赖雌激素和生长因子的人乳腺癌和乳腺上皮细胞增殖中的作用。AKT1的活性将通过免疫复合物激酶测定，水平将通过核糖核酸酶保护试验和Western blotting来确定。AKT1的活性将与雌激素(在ER+细胞中)和生长因子(例如：雌激素+细胞)的增殖反应有关。IGF-I)。不同的AKT亚型的作用也将通过显性阴性的AKT逆转录病毒和异构体特异性的AKT反义寡核苷酸(AON)来评估。2)确定AKT1在阻止乳腺癌和乳腺上皮细胞凋亡中的作用。AKT1在细胞凋亡中的作用将通过显性-阴性形式的AKT1或同工型特异性的AKT AON来评估。细胞凋亡的特征是DNA断裂的原位末端标记和caspase-3的激活。抑制AKT1引起的基因表达变化将通过基因芯片寡核苷酸微阵列分析和消减杂交进行评估。通过检测显性阴性AKT1和AKT AON转导的乳腺癌细胞中bcl2家族成员的磷酸化程度来评价AKT1在防止细胞凋亡中的翻译后效应。3)确定乳腺导向表达AKT1和GAG-AKT1转基因是否会导致增生和转化。在MMTV LTR的控制下，将产生在乳腺中表达AKT1或其固有活性的Gag-AKT1癌基因的转基因小鼠。野生型和转基因小鼠的乳腺组织中的基因表达将通过基因芯片和消减杂交分析进行评估。来自转基因动物的乳腺上皮和肿瘤的原代培养将表现为依赖生长因子的增殖、对凋亡的敏感性、软琼脂中的锚定非定植生长和在裸鼠体内的致瘤性。

This proposal will address the hypothesis that AKT1, the cellular homolog of the protein-serine/threonine kinase oncogene v-akt, plays a key role in the proliferation, survival and etiology of breast cancer. This will be addressed by the following Specific Aims: 1) Determine the role of AKT1, and possibly other AKT isoforms, in estrogen- and growth factor-dependent proliferation of human breast cancer and breast epithelial cells. AKT1 activity will be quantified by immunocomplex kinase assays and levels will be determined by RNase protection assay and western blotting. AKT1 activity will be correlated to proliferation in response to estradiol (in ER+ cells) and growth factors (eg. IGF-I). The role of different AKT isoforms will also be assessed with dominant-negative AKT retroviruses and isoform-specific AKT antisense oligonucleotides (AONs). 2) Determine the role of AKT1 in preventing apoptosis in breast cancer and breast epithelial cells. The role of AKT1 in apoptosis will be evaluated with dominant-negative forms of AKT1 or isoform-specific AKT AONs. Apoptosis will be characterized by in situ end-labeling of DNA breaks, and caspase-3 activation. Changes in gene expression resulting from inhibition of AKT1 will be assessed by GeneChip Oligonucleotide microarray analysis and subtractive hybridization. The post-translational effects of AKT1 in preventing apoptosis will be evaluated by determining the phosphorylation of bcl-2 family members in breast cancer cells transduced with a dominant-negative AKT1 and AKT AONs. 3) Determine if mammary gland-directed expression of the AKT1 and Gag-akt1 transgenes results in hyperplasia and transformation. Transgenic mice will be generated that express AKT1 or the constitutively active Gag-akt1 oncogene in the mammary gland under the control of the MMTV LTR. Gene expression in mammary tissue from wild-type and transgenic mice will be assessed by GeneChip and subtractive hybridization analyses. Primary cultures of mammary epithelium and tumors arising from transgenic animals will be characterized for growth factor-dependent proliferation, susceptibility to apoptosis, anchorage-independent growth in soft agar and tumorigenicity in nude mice.