目的:蛋氨酸成瘾是癌细胞的一般和基本标志,被称为霍夫曼效应。以前,Vanhamme和Szpirer表明,可以通过将激活的HRAS1基因转染到正常细胞系中来诱导蛋氨酸成瘾。在本研究中,我们调查了c-MYC癌基因在癌症蛋氨酸成瘾中的作用,通过比较c-Myc表达和甲硫氨酸成瘾的骨肉瘤细胞和罕见的甲硫氨酸非依赖性回复体的恶性,来源于蛋氨酸成瘾细胞.
方法:蛋氨酸非依赖性回复性143B骨肉瘤细胞(143B-R)来源于蛋氨酸成瘾的亲本143B骨肉瘤细胞(143B-P),通过在重组蛋氨酸酶耗尽蛋氨酸的培养基中连续培养。比较蛋氨酸成瘾亲本细胞和蛋氨酸非依赖性回复体细胞的体外恶性程度。进行了以下实验:对于143B-P和143B-R细胞,细胞增殖能力用细胞计数测定,在塑料和软琼脂上确定菌落形成能力,全部在含蛋氨酸的Dulbecco改良鹰培养基(DMEM)中。在原位异种移植裸鼠模型中测量肿瘤生长,比较143B-P和143B-R细胞的体内恶性程度。用蛋白质免疫印迹法检测c-MYC表达,并在143B-P和143B-R细胞中进行比较。
结果:143B-R细胞增殖能力降低,与143B-P细胞相比,在含蛋氨酸的培养基中(p=0.003)。143B-R细胞在塑料(p=0.003)和软琼脂上的集落形成能力降低,与含蛋氨酸培养基中的143B-P细胞相比。143B-R细胞在原位异种移植裸鼠模型中肿瘤生长减少,与143B-P细胞相比,(p=0.002)。这些结果表明143B-R不依赖甲硫氨酸的回复体细胞失去了恶性。c-MYC在143B-R蛋氨酸非依赖性回复性骨肉瘤细胞中的表达降低,与143B-P细胞相比,(p=0.0007)。
结论:本研究表明c-MYC表达与癌细胞的恶性肿瘤和蛋氨酸成瘾有关。目前对c-MYC的研究,和先前对HRAS1的研究表明,癌基因可能在蛋氨酸成瘾中起作用,这是所有癌症的标志,以及恶性肿瘤。
OBJECTIVE: Methionine addiction is a general and fundamental hallmark of cancer cells, termed the Hoffman effect. Previously Vanhamme and Szpirer showed that methionine addiction could be induced by transfection of the activated HRAS1 gene to a normal cell line. In the present study, we investigated the role of the c-MYC oncogene in methionine addiction of cancer, by comparison of c-Myc expression and malignancy of methionine-addicted osteosarcoma cells and rare methionine-independent revertants, derived from the methionine-addicted cells.
METHODS: Methionine-independent revertant 143B osteosarcoma cells (143B-R) were derived from methionine-addicted parental 143B osteosarcoma cells (143B-P), by continuous culture in medium depleted of methionine by recombinant methioninase. To compare in vitro malignancy of methionine-addicted parental cells and methionine-independent revertant cells, the following experiments were performed: for 143B-P and 143B-R cells, cell proliferation capacity was measured with a cell-counting assay, and colony-formation capacity was determined on plastic and in soft agar, all in methionine-containing Dulbecco\'s Modified Eagle\'s Medium (DMEM). Tumor growth was measured in orthotopic xenograft nude-mouse models, to compare in vivo malignancy of 143B-P and 143B-R cells. c-MYC expression was examined with western immunoblotting and compared in 143B-P and 143B-R cells.
RESULTS: 143B-R cells had reduced cell proliferation capacity, compared to 143B-P cells, in methionine-containing medium (p=0.003). 143B-R cells had reduced colony formation capacity on plastic (p=0.003) and in soft agar, compared to 143B-P cells in methionine-containing medium. 143B-R cells had reduced tumor growth in orthotopic xenograft nude-mouse models, compared to 143B-P cells, (p=0.002). These results demonstrate that 143B-R methionine-independent revertant cells lost malignancy. Expression of c-MYC was reduced in 143B-R methionine-independent revertant osteosarcoma cells, compared to 143B-P cells, (p=0.0007).
CONCLUSIONS: The present study demonstrated that c-MYC expression is linked to malignancy and methionine addiction of cancer cells. The present study on c-MYC, and the previous study on HRAS1, suggest that oncogenes may play a role in methionine addiction, which is a hallmark of all cancers, as well as in malignancy.