Rosenberg C D, Ferrone S, Hamby C V, Mancino V, Graf L H
Cornell University Graduate School of Medical Sciences, New York, New York 10021.
Cancer Res. 1990 Mar 1;50(5):1559-65.
The gene that encodes the membrane-bound Mr 100,000 human melanoma-associated antigen (MAA) defined by mouse mAb 376.96, a leukocyte and fibroblast interferon-modulated glycoprotein having preferential distribution on melanoma and carcinoma cells, has been transfected into the mouse melanoma cell line B78H1 as a step toward molecular cloning and characterization of the MAA. Primary, secondary, and tertiary B78H1 transfectants expressing the Mr 100,000 MAA gene were generated by treatment with coprecipitated DNA from Mr 100,000 MAA+ human or transfectant mouse cells and they were detected by an indirect RBC rosetting assay. The Mr 100,000 MAA gene was also transferred into K-1735 mouse melanoma cells and into nonmalignant and malignant mouse fibroblast lines. The species immunoprecipitated by mAb 376.96 from human melanoma cells (Mr 100,000) and from mouse melanoma transfectant cells (Mr 97,000-100,000) were both converted to molecule(s) having an Mr of approximately 70,000 by enzymatic removal of asparagine-linked carbohydrate residues. Two independent secondary transformant clones of B78H1 cells express Mr 100,000 MAA antigenicity at levels significantly higher than those observed when one or two copies of the gene are present. Clone Mr 100,000 secondary-A spontaneously overexpresses Mr 100,000 MAA at least 5-fold and has greater than or equal to 10 times elevated levels of putatively Mr 100,000 MAA gene-associated human alu family repeat element (h-alu)-positive restriction fragments relative to "single" copy secondary transfectant cells. Clone Mr 100,000 secondary-B has increased copy number and expression of Mr 100,000 MAA as a consequence of a selective co-amplification procedure which is targeted to a mouse wild type dihydrofolate reductase (dhfr) gene expression vector. This vector was co-introduced into B78H1 cells in addition to the DNA of Mr 100,000 MAA+ primary transfectant cells and the initially selected aminoglycoside phosphotransferase (neo) gene vector. Stepwise selections of a secondary Mr 100,000 MAA+ transfectant clone with increasing concentrations of the dihydrofolate reductase-inhibitory antimetabolite methotrexate led to progressive increases in copy numbers of the introduced dhfr gene and to parallel increases in h-alu sequences, in cellular levels of dihydrofolate reductase protein, and in cellular mAb 376.96 reactivity. Levels of these entities ultimately reached 50-fold, relative to levels expressed prior to amplification. The array of h-alu+ restriction fragments amplified in Mr 100,000 secondary-B cell DNA is very similar to that observed in Mr 100,000 secondary-A cell DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
由小鼠单克隆抗体376.96所定义的编码膜结合型分子量为100,000的人黑色素瘤相关抗原（MAA）的基因，是一种白细胞和成纤维细胞干扰素调节的糖蛋白，在黑色素瘤和癌细胞上有优先分布，已被转染到小鼠黑色素瘤细胞系B78H1中，作为迈向MAA分子克隆和特性分析的一步。通过用来自分子量为100,000的MAA+人或转染小鼠细胞的共沉淀DNA处理，产生了表达分子量为100,000的MAA基因的一级、二级和三级B78H1转染子，并通过间接红细胞花环试验进行检测。分子量为100,000的MAA基因也被转入K-1735小鼠黑色素瘤细胞以及非恶性和恶性小鼠成纤维细胞系中。单克隆抗体376.96从人黑色素瘤细胞（分子量为100,000）和小鼠黑色素瘤转染细胞（分子量为97,000 - 100,000）中免疫沉淀的物种，通过酶促去除天冬酰胺连接的碳水化合物残基后，都转化为分子量约为70,000的分子。两个独立的B78H1细胞二级转化克隆表达分子量为100,000的MAA抗原性的水平，显著高于基因拷贝数为一或两个时所观察到的水平。克隆分子量为100,000二级-A自发地过量表达分子量为100,000的MAA至少5倍，相对于“单拷贝”二级转染细胞，推定的分子量为100,000的MAA基因相关的人alu家族重复元件（h-alu）阳性限制性片段水平升高了10倍或更多。克隆分子量为100,000二级-B由于针对小鼠野生型二氢叶酸还原酶（dhfr）基因表达载体的选择性共扩增程序，增加了分子量为100,000的MAA的拷贝数和表达。除了分子量为100,000的MAA+一级转染细胞的DNA和最初选择的氨基糖苷磷酸转移酶（neo）基因载体外，该载体还被共导入B78H1细胞中。用浓度递增的二氢叶酸还原酶抑制性抗代谢物甲氨蝶呤对二级分子量为100,000的MAA+转染克隆进行逐步选择，导致导入的dhfr基因拷贝数逐渐增加，h-alu序列、细胞中二氢叶酸还原酶蛋白水平以及细胞对单克隆抗体376.96的反应性也平行增加。相对于扩增前表达的水平，这些实体的水平最终达到了50倍。在分子量为100,000二级-B细胞DNA中扩增的h-alu+限制性片段阵列与在分子量为100,000二级-A细胞DNA中观察到的非常相似。（摘要截短至400字）
