AKT PROTOONCOGENE IN BREAST CANCER

该提案将解决的假设，AKT 1，蛋白质丝氨酸/苏氨酸激酶癌基因v-akt的细胞同源物，在乳腺癌的增殖，生存和病因中起着关键作用。 这将通过以下具体目的来解决：1）确定AKT 1和可能的其他AKT同种型在人乳腺癌和乳腺上皮细胞的雌激素和生长因子依赖性增殖中的作用。 AKT 1活性将通过免疫复合物激酶测定法定量，水平将通过RNA酶保护测定法和蛋白质印迹法测定。 AKT 1活性将与响应雌二醇（ER+细胞）和生长因子（例如，IGF-I）。 还将使用显性阴性AKT逆转录病毒和亚型特异性AKT反义寡核苷酸（AON）评估不同AKT亚型的作用。 2)确定AKT 1在预防乳腺癌和乳腺上皮细胞凋亡中的作用。 AKT 1在细胞凋亡中的作用将用显性阴性形式的AKT 1或同种型特异性AKT AON进行评价。细胞凋亡的特征在于DNA断裂的原位末端标记和半胱天冬酶-3活化。 通过基因芯片寡核苷酸微阵列分析和消减杂交评估AKT 1抑制导致的基因表达变化。 将通过测定用显性阴性AKT 1和AKT AON转导的乳腺癌细胞中bcl-2家族成员的磷酸化来评价AKT 1在防止细胞凋亡中的翻译后作用。 3)确定乳腺定向表达的AKT 1和Gag-akt 1转基因是否导致增生和转化。将产生在MMTV LTR控制下在乳腺中表达AKT 1或组成型活性Gag-akt 1癌基因的转基因小鼠。 将通过基因芯片和消减杂交分析评估野生型和转基因小鼠乳腺组织中的基因表达。 将对转基因动物产生的乳腺上皮和肿瘤的原代培养物进行生长因子依赖性增殖、细胞凋亡易感性、软琼脂中的锚定非依赖性生长和裸鼠中的致瘤性表征。

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.