Wrathall J R, Oliver C, Silagi S, Essner E
J Cell Biol. 1973 May;57(2):406-23. doi: 10.1083/jcb.57.2.406.
Low concentrations (1-3 microg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B(5)59) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C(3)471), derived by long-term culture of B(5)59 cells in 1 microg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B(5)59 cells during a 7-day period of growth with BrdU (3 microg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T(1) and T(2) forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T(1)) was consistently affected earlier than the other (T(2)). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.
低浓度（1 - 3微克/毫升）的5 - 溴脱氧尿苷（BrdU）可可逆性抑制培养的B16小鼠黑色素瘤细胞的一个高色素克隆（B(5)59）的色素沉着。我们发现，通过在1微克/毫升的BrdU中长期培养B(5)59细胞获得的无色素细胞（克隆C(3)471）完全无黑色素，没有生化或细胞化学可检测到的酪氨酸酶活性，也没有前黑素小体的超微结构证据。在添加BrdU（3微克/毫升）的7天生长期间，研究了B(5)59细胞中色素沉着被抑制的过程。酪氨酸酶活性测定表明，活性在1天后降低，并逐渐下降，到7天时接近零。这种降低的一小部分直接归因于一种可透析的酪氨酸酶活性抑制剂的出现。丙烯酰胺凝胶电泳显示两条活性带，其相对迁移率（Rx值）与可溶性酪氨酸酶的T(1)和T(2)形式相对应。在添加BrdU生长期间，两者均逐渐减少，但一种形式（T(1)）始终比另一种形式（T(2)）更早受到影响。超微结构细胞化学研究还显示对酪氨酸酶反应产物的定位有早期影响。在第3天，反应产物不再存在于高尔基囊泡和与高尔基体相关的光滑表面小管中，但仍可见于前黑素小体、复合黑素小体以及偶尔与高尔基体相关的囊泡内。到第7天，酪氨酸酶反应产物通常无法检测到。在添加BrdU生长期间，前黑素小体的数量逐渐减少。前黑素小体集中在近核区域，在第3天，许多前黑素小体包含在异常大且数量众多的复合黑素小体中。在第7天时，很少见到前黑素小体和复合黑素小体，此时培养物几乎无黑色素。BrdU对黑色素生成的协同抑制作用可能为研究这一过程的正常调节提供一个有用的模型。
