CHARACTERIZATION OF MELANOMA GROWTH ACTIVITY

研究的重点是内生增长的特征 恶性黑色素瘤细胞产生的因子和描述 这种生长因子在癌前痣中的早期表达。 一个 已经分离出黑素细胞的自刺激生长因子， 部分特征。 这种黑色素瘤生长刺激活性（MGSA） 存在于抗原相关的酸和热稳定的 这些多肽似乎不同于先前描述的其它多肽， 生长因子 MGSA在染色体正常、不活动的 痣，但痣表现出染色体异常是MGSA阳性。 MGSA可以从冻干培养物的乙酸提取物中纯化 通过Hs 0294人黑素瘤细胞系调节的培养基。 当这个 对提取物进行分子筛色谱、RP-HPLC和 制备凝胶电泳，低（大于14- 16 Kd）和高 可以分离（24- 26 Kd）分子量形式的MGSA。 MGSA是 可与125 I-EGF竞争I类TGF活性分离， 由这一细胞系产生。 MGSA还可以通过免疫亲和层析法纯化。 使用MGSA的单克隆抗体进行层析，然后进行凝胶渗透色谱。 过滤HPLS。 当35 S-蛋氨酸标记的免疫沉淀物 对Hs 0294细胞的提取物进行还原性SDS-PAGE， 放射自显影，主要标记带的Mr大于20 Kd。 现在的努力将指向：（1）确定关系 低于MGSA的低和高分子量形式之间， 开发一个体外翻译系统，以确定 MGSA的前体形式：（2）开发MGSA的放射受体测定法; (MGSA将通过上述方法纯化，使用碘化 Bolton-Hunter试剂和结合试验将使用Hs 0294和 NRK细胞）（3）表征分离的质膜中的MGSA受体 使用125 I-MGSA和双功能交联剂的制备， 辛二酸二琥珀酰亚胺酯;（4）比较 生物活性和免疫反应性MGSA和MGSA受体的分布， 正常痣、恶性黑色素瘤和非恶性和非黑色素瘤 恶性控制。 将培养痣、黑色素瘤和其他肿瘤细胞 在体外，MGSA产生将通过免疫组织化学测定， 生物活性测定和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的结合与生物学应答。

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.