Ganapathi R, Grabowski D, Schmidt H, Bell D, Melia M
Cancer Res. 1987 Jul 1;47(13):3464-8.
Adriamycin (ADR)-resistant sublines of B16-BL6 mouse melanoma selected by exposure to increasing concentrations of ADR were characterized in vitro for growth properties and in vivo for tumorigenicity and pulmonary metastases. The progressively resistant sublines adapted to grow in the presence of 0.025, 0.05, 0.1, and 0.25 microgram/ml ADR in monolayer culture were found to be 5-, 10-, 20-, and 40-fold ADR-resistant, respectively, compared to the parental sensitive cells, using a soft-agar colony assay and continuous ADR treatment for 7 days. The doubling time in monolayer culture of the parent sensitive and progressively ADR-resistant sublines of B16-BL6 melanoma cells was approximately 16-18 h. Although the colony-forming efficiency in soft agar of parental sensitive cells was only 0.5-4%, the 5-, 10-, 20-, and 40-fold ADR-resistant sublines had colony-forming efficiencies of 15, 20, 30, and 77%, respectively. Tumorigenicity in C57BL/6 mice of progressively ADR-resistant sublines was similar to parental sensitive cells following s.c. and i.p. implantation of 10(5)-10(6) tumor cells. Experimental pulmonary metastases were significantly lower in ADR-resistant sublines with progressive resistance. Additionally, unlike the parental sensitive and 5-fold ADR-resistant B16-BL6 cells, the 10-, 20-, and 40-fold ADR-resistant sublines were spontaneously nonmetastatic. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical detection of P-glycoprotein revealed the presence of a Mr 170,000 plasma membrane glycoprotein in the 40-fold ADR-resistant subline and its counterpart maintained for 1 year in ADR-free medium. Results from this study suggest that progressively ADR-resistant B16-BL6 mouse melanoma cells selected in vitro demonstrate a marked increase in colony formation in soft agar and a decrease in the ability to produce pulmonary metastases, without alterations in tumorigenicity.
通过暴露于浓度不断增加的阿霉素（ADR）来筛选出B16 - BL6小鼠黑色素瘤的阿霉素耐药亚系，并在体外对其生长特性进行表征，在体内对其致瘤性和肺转移情况进行研究。采用软琼脂集落测定法并连续用阿霉素处理7天，发现在单层培养中能适应在0.025、0.05、0.1和0.25微克/毫升阿霉素存在下生长的逐渐耐药亚系，与亲本敏感细胞相比，分别具有5倍、10倍、20倍和40倍的阿霉素耐药性。B16 - BL6黑色素瘤细胞的亲本敏感细胞和逐渐产生阿霉素耐药性的亚系在单层培养中的倍增时间约为16 - 18小时。虽然亲本敏感细胞在软琼脂中的集落形成效率仅为0.5 - 4%，但具有5倍、10倍、20倍和40倍阿霉素耐药性的亚系的集落形成效率分别为15%、20%、30%和77%。在皮下和腹腔注射10⁵ - 10⁶个肿瘤细胞后，逐渐产生阿霉素耐药性的亚系在C₅₇BL/6小鼠中的致瘤性与亲本敏感细胞相似。具有逐渐耐药性的阿霉素耐药亚系的实验性肺转移明显较低。此外，与亲本敏感细胞和具有5倍阿霉素耐药性的B16 - BL6细胞不同，具有10倍、20倍和40倍阿霉素耐药性的亚系自发不发生转移。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和P - 糖蛋白的免疫化学检测显示，在具有40倍阿霉素耐药性的亚系及其在无阿霉素培养基中维持1年的对应亚系中存在一种分子量为170,000的质膜糖蛋白。本研究结果表明，体外筛选出的逐渐产生阿霉素耐药性的B16 - BL6小鼠黑色素瘤细胞在软琼脂中的集落形成显著增加，产生肺转移的能力下降，而致瘤性无改变。
