CHARACTERIZATION OF MELANOMA GROWTH ACTIVITY

调查的重点是内生增长的特征。 恶性黑色素瘤细胞产生的因子及其对 该生长因子在癌前痣中的早期表达。一个 黑素细胞的自刺激性生长因子已被分离并 有部分特征的。这种黑色素瘤生长刺激活性(MGSA) 存在于一个抗原性相关的家族，酸和热稳定 似乎与先前描述的其他多肽不同的多肽 增长因素。MGSA在染色体正常、不活跃的患者中检测不到 但表现为染色体异常的痣均为MGSA阳性。 MGSA可以从冻干培养物的醋酸提取物中得到纯化 Hs0294人黑色素瘤细胞株条件培养液。当这件事 提取物经分子筛层析、反相高效液相色谱(RP-HPL C)和 制备性凝胶电泳法，低(大于14-16Kd)和高 (24-26Kd)分子量形式的MGSA可以被分离出来。MGSA是 可与125I-EGF竞争的I类转化生长因子活性分开，这也是 由这种细胞系产生。MGSA也可以通过免疫亲和来纯化 用抗MGSA的单抗进行层析，然后用凝胶 过滤HPLS。当从35S-蛋氨酸标记的免疫沉淀 对Hs0294细胞提取液进行还原SDS-PAGE，然后 放射自显影，主要标记条带MR>20Kd。 现在的努力方向是：(1)确定关系 在以下低分子形式和高分子形式的MGSA和 开发一种体外翻译系统，以确定 MGSA的前体形式；(2)建立MGSA的放射受体分析方法； (MGSA将通过上述方法提纯，使用碘 Bolton-Hunter试剂和结合分析将使用Hs0294和 NRK细胞)(3)分离质膜MGSA受体的特征 使用125I-MGSA和双官能交联剂的制剂， 琥珀酸二丁二酰亚胺；(4)比较不同浓度丁二酰亚胺的细胞分布 MGSA生物活性和免疫反应及MGSA受体在脑内的分布 正常痣、恶性黑色素瘤、非恶性和非黑色素瘤 恶性对照。痣、黑色素瘤和其他肿瘤细胞将被培养 在体外，MGSA的产生将通过免疫组织化学和 生物活性分析和MGSA受体分布将使用 MGSA-放射受体测定。将对MGSA进行比较 在这些培养物中，对MGSA的结合与生物反应。

The investigations focus on the characterization of an endogenous growth factor produced by malignant melanoma cells and the description of the early expression of this growth factor in premalignant nevi. An autostimulatory growth factor for melanocytes has been isolated and partially characterized. This melanoma growth stimulatory activity (MGSA) resides in a family of antigenically related, acid and heat stable polypeptides which appear to be different from other previously described growth factors. MGSA is not detectable in chromosomally normal, inactive nevi, but nevi exhibiting chromosomal abnormalities are MGSA positive. MGSA can be purified from acetic acid extracts of lyophilized culture medium conditioned by the Hs0294 human melanoma cell line. When this extract is subjected to molecular sieve chromatography, RP-HPLC and preparative gel electrophoresis, low (greater than 14-16Kd) and high (24-26Kd) molecular weight forms of MGSA can be isolated. MGSA is separable from the 125I-EGF competing Class I TGF activity which is also produced by this cell line. MGSA can also be purified by immunoaffinity chromatography using a monoclonal antibody to MGSA, followed by gel filtration HPLS. When immunoprecipitates from 35S-methionine labeled extracts of Hs0294 cells were subjected to reducing SDS-PAGE followed by autoradiography, the major labeled bands had a Mr of greater than 20Kd. Efforts will now be directed toward: (1) determining the relationship between below the low and high molecular weight forms of MGSA and developing an invitro translation system to determine the nature of the precursor form of MGSA; (2) developing a radioreceptor assay for MGSA; (MGSA will be purified by the methods described above, iodinated using Bolton-Hunter reagent and binding assays will be developed using Hs0294 and NRK cells) (3) characterizing the MGSA receptor in isolated plasma membrane preparations using 125I-MGSA and the bifunctional cross-linking reagent, disuccinimidyl suberate; (4) comparing the cellular distribution of bioactive and immunoreactive MGSA and the distribution of MGSA receptors in normal nevi, malignant melanomas, and non-malignant and non-melanoma malignant controls. Nevus, melanoma and other tumor cells will be cultured in vitro, MGSA production will be determined by immunohistochemical and bioactivity assays, and MGSA receptor distribution will be evaluated using the MGSA-radioreceptor assay. Comparisons will be made regarding MGSA binding versus biological response to MGSA in these cultures.