Abstract
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.
Highlights
Low concentrations (1-3 μg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B 5 59) of cultured B16 mouse melanoma cells
Tyrosinase activity was cytochemically demonstrable within GERL and small vesicles near the Golgi apparatus, melanin was not evident in these structures [16, 17]
Fig . 4 depicts the progressive reduction of T1 and T2 during treatment with 3 ug of BrdU/ml. In this experiment 7-day treated cells had no detectable activity by assay, and no bands of tyrosinase activity were seen upon electrophoresis
Summary
The melanoma cell line was derived from the B16 mouse melanoma by Hu [21]. Pigmented clones were produced from this line by Silagi [22] and the highly melanotic clone, B559, was used in this investigation. For demonstration of tyrosinase activity the formaldehyde-fixed cells were rinsed in buffer and directly incubated in DOPA-containing medium [16, 30] while glutaraldehyde-fixed cells were rinsed overnight in buffer, containing 7% sucrose, before incubation. Monolayer cultures of cells were rinsed in physiological saline, fixed for 1 h at 4°C in 3% buffered glutaraldehyde, rinsed in buffer, and left overnight at 4°C in buffer containing 7% sucrose. $ Replicate cultures of B 559 cells were grown for 3 days in regular medium (Control) or medium containing 3 .sg of BrdU/ml (BrdU-treated). Other cells were left overnight at 4°C in 0.2 M cacodylate buffer containing 10-2 M Na diethyldithiocarbamate (DECA, a copper chelator), 2 mg DOPA/ml and 7 0/0 sucrose, rinsed thoroughly in buffer, and incubated with DOPA. The DECA-treated cells were rinsed in buffer, left for 1 h in 1 0/0 aqueous copper sulfate, and rinsed again in buffer before incubation
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