HCGB FROM CERVICAL CANCER: PEPTIDE HETEROGENITY

Dot和CaSki人癌细胞异位分泌hCG-β 包含两个组分，异位hCG-β-I和异位hCG-β-II。 异位hCG-β-II缺乏C端肽(CTP)，基于缺乏 被CTP抗体识别，缺乏易感性 嗜热溶血素(通常切割CTP)，以及缺乏结合 花生凝集素(AH)的去分析衍生物(专用于 CTP上O-连接低聚糖的Gal-GalNAc)。免疫反应阳性 18例中有13例的血清或尿液中检测到类似人绒毛膜促性腺激素的物质 宫颈癌患者。大型滚筒瓶的使用条件 对CaSki宫颈癌细胞的培养进行了优化。一种方法，用于 异位hCG-β-I和hCG-β-II的分离已经得到验证， 涉及差示亲和层析：(1)\抗hCG-α 琼脂糖基；(2)反[CTP导向]hCG-β-琼脂糖基； (3)反义[核心导向]hCG-β琼脂糖凝胶。异位hCG-β-II被洗脱 从第三阶段开始，用4M盐酸胍，pH为3，脱盐。这个 纯化的异位hCG-β-II未能与标准hCG-α结合 在标准hCG-β和标准hCG-α结合的条件下。 获得的证据表明，食物中含有额外的碳水化合物 异位hCG-β。含免疫活性物质的DOT和CaSki培养液 明显比标准hCG-β更大的类似hCG-β的成分 (取自妊娠尿hCG)，经凝胶过滤和钠盐过滤证实 十二烷基硫酸酯凝胶电泳法(后者在孵育后 含有放射性氨基酸的CaSki细胞)。(C)

Ectopic hCG-beta secreted by DoT and CaSki human carcinoma cell lines contains two components, ectopic hCG-beta-I and ectopic hCG-beta-II. Ectopic hCG-beta-II lacks the C-terminal peptide (CTP), based on lack of recognition by antibodies against CTP, lack of susceptibility to thermolysin (which normally cleaves the CTP), and lack of binding of the desialylated derivative to Arachis hypogaea (AH) lectin (specific for Gal-GalNAc of the O-linked oligosaccharides on the CTP). Immunoreactive hCG-beta-like material has been detected in the serum or urine of 13 of 18 patients with cervical carcinoma. Conditions for large-scale roller bottle culture of CaSki cervical carcinoma cells were optimized. A method to separate ectopic hCG-beta-I from ectopic hCG-beta-II has been validated and involves differential affinity chromatography with: (1)\anti-hCG-alpha Sepharose; (2)\anti-[CTP-directed]hCG-beta-Sepharose; and (3)\anti-[core-directed]hCG-beta Sepharose. Ectopic hCG-beta-II is eluted from the third stage with 4 M guanidine\HC1, pH 3, and desalted. The purified ectopic hCG-beta-II failed to combine with standard hCG-alpha under conditions where standard hCG-beta combined with standard hCG-alpha. Evidence was obtained for the presence of additional carbohydrates on ectopic hCG-beta. DoT and CaSki culture fluids contained immunoreactive hCG-beta-like components of apparently larger size than standard hCG-beta (from pregnancy urine hCG) as demonstrated by gel filtration and sodium dodecyl sulfate-gel electrophoresis (the latter following incubation of CaSki cells with radioactive amino acids). (C)