A Study of the Mechanism of Action and Role of a Natural Inhibitor of Dopa Autoxidation isolated from Guinea Pig Skin**From the Skin Biochemistry Section, Unilever Research Laboratory, Isleworth Middlesex, EngLand.

Flawn, Phyllis C.; Biol, M. I.; Wilde, Peter F.

doi:10.1111/1523-1747.ep12259363

Cited by 6 publications

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“…Completely amelanotic cells grow in a fibroblastic manner . X 180. of tyrosinase have previously been reported in extracts of mammalian skin (33)(34)(35)(36), melanomas (37)(38)(39)(40), amelanotic melanoma cells in culture (22), and amelanotic cells resulting from the hybridization of pigmented melanoma cells to L cells (41) . Our preliminary characterization of the inhibitory material in C3471 cells indicates the presence of heat-stable, dialyzable material capable of inhibiting both DOPA autoxidation and tyrosinase-catalyzed production of dopachrome .…”

Section: Discussionmentioning

confidence: 97%

SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE

Wrathall

Oliver

Silagi

et al. 1973

The Journal of Cell Biology

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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 . We have found that unpigmented cells (clone C 3 471), derived by long-term culture of B 5 59 cells in 1 ug 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 559 cells during a 7-day period of growth with BrdU (3 µg/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, and T2 forms of soluble tyrosinase . Both were progressively reduced during growth with BrdU but one form (T 1 ) was consistently affected earlier than the other (T2 ) . 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 . 0 6Low concentrations of 5-bromodeoxyuridine (BrdU) have been shown to suppress differentiated functions preferentially in a variety of cell types (1-9) . Silagi and Bruce (5) reported that treatment of melanotic melanoma cells (clone B559) with BrdU altered cellular morphology and suppressed both pigmentation and tumorigenicity while cell proliferation continued at a near normal level . These effects appeared dependent upon incorporation of the BrdU into cellular DNA as an analogue of thymidine . The effects upon morphology, pigmentation, and tumorigenicity were reversible upon growth of the cells in normal medium (5, 10) . The molecular basis of this reversible effect is

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Section: Discussionmentioning

confidence: 97%

SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE

Wrathall

Oliver

Silagi

et al. 1973

The Journal of Cell Biology

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“…In addition, it is known that the DOPA oxidase reaction in the L-B method can result in non-specific false-positive reactions due to auto-oxidation and/or internal peroxidase [10,11]. False-positive reactions with other oxidations were also observed in the other classical DOPA oxidase reactions.…”

Section: Discussionmentioning

confidence: 99%

New Methodological Approach for the Rapid and Sensitive Detection of Melanocytes and Melanocytic Tumours

et al. 2009

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Background: During melanogenesis, the activity of tyrosinase (the key enzyme for melanin synthesis) is important in the diagnosis of melanomas. Methods: Nine samples were included in this study, based on the availability of four malignant melanomas and five benign tumours. Our new approach is based on a combination of the DOPA oxidase and the glyoxylic acid (GA) methods for catecholamine (named the ‘DOPA-GA method’). Here, we compared the conventional DOPA oxidase method with that of the DOPA-GA method. Results: In all malignant melanomas, the fluorescence intensity of the DOPA-GA method was strongly expressed within the cytoplasm. The melanoma cells showed an obvious sensitivity compared with the Laidlaw and Blackberg method, as did the melanocytes in the normal skin. Detection was easier with the DOPA-GA method than the Laidlaw and Blackberg method. Hence, we developed a sensitive and rapid method with a total assay time of less than 30 min. Conclusion: DOPA-GA reflects tyrosinase activity in melanocytic tumours, and our new approach is an improvement on the conventional DOPA oxidase method, with regard to both specificity and sensitivity. The DOPA-GA method will be useful for routine pathological testing and melanogenetic studies of melanocytes and melanocytic tumours.

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