False Dopa Reaction in Studies of Mammalian Tyrosinase: Some Characteristics and Precautions

White, Roger; Hu, Funan; Roman, Nickolas

doi:10.3109/10520298309066744

Cited by 17 publications

(6 citation statements)

References 7 publications

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“…Optimal cell growth in culture was obtained with a mixture containing equal amounts of DMEM and Ham's F10 culture medium; growth in RPMI-1640 medium was inferior and MEM supported cell proliferation only poorly (results not shown). To ascertain whether our cultures contained melanin synthesizing cells, a cytochemical assay for tyrosinase was performed (22). All cultures clearly showed tyrosinase activity (results not shown).…”

Section: Resultsmentioning

confidence: 99%

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Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases

et al. 1995

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Continuously growing cell cultures, testing positive for tyrosinase activity, were derived from two brain and three lymph-node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation fate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 f 130 p M BrdUrd (range:130-520; n = 11) vs. 650 f 50 pM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 2 55 pM (range: 65-400 FM) for 20% oxygen vs. 360 f 130 p M BrdUrd for 5% oxygen. The cell cycle kinetic mechanism of BrdUrd-induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3-fold variation in BrdUrd sensitivity. I n one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 3325Wethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy. Key terms: Bromodeoxyuridine labeling, melanoma cells, metastasis, Hoechst 33258 dye, ethidium bromide, bivariate flow cytometry, G2 phase, tyrosinaseThe growing incidence of malignant melanoma is becoming a major public health concern ( 11,17,20). Notwithstanding efficient surgical treatment of primary skin tumors, the propensity of malignant melanoma to form metastases contributes to a considerable extent to melanoma morbidity (5). Melanoma metastases present with considerable heterogeneity, which makes them a difficult target for radio-and chemotherapy (1,3,6).We use a highly sensitive cell cycle kinetic assay which allows the systematic study of activated tumor cells with respect to sensitivities toward chemotherapeutic agents ( 13). The assay involves exposure of cell cultures to 50-100 pM concentrations of bromodeoxyuridine (RrdIJrd) for 72 h. Multiple rounds of DNA replication are subsequently detected by staining cells with Hoechst 33258 and ethidium bromide and analysis in a flow cytometer equipped with ultraviolet (UV) excitation ( 1 5,16). In contrast to most other types of cells, melanoma cells were found to be very sensitive to BrdUrd. The availability of several growing cell cultures from each tumor biopsy permitted the study within a single tumor of putative heterogeneity of response toward a cytotoxic drug. MATERIALS AND METHODS Establishment of Melanoma CulturesTwo brain and three lymph-node melanoma metastases were obtained from five different patients undergoing surgical resection. Tumors were transported in sterile culture media and maintained at room temperature. After rinsing with culture media, consisting of an equal mixture of Dulbecco's modificatio...

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Section: Resultsmentioning

confidence: 99%

“…Pigment production was monitored by the procedure of White et al (22). Briefly, cells were seeded onto slides and cultured in DMEM/F10 media for 24-48 h. Subsequently, slides were rinsed 3 times with PBS and cells were fixed for 2 0 min at room temperature in 10% formalin in PBS.…”

Section: Tyrosinase Assay and Cytogenetic Evaluationmentioning

confidence: 99%

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases

et al. 1995

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“…The definition used here will be that of Hirobe, namely: melanoblasts may have premelanosomes (melanosome precursors lacking melanin), but contain no tyrosinase activity (18). This can be ascertained by a histochemical test for tyrosinase, the formation of a brown to black product from L-dopa (18,19). There is also an internediate cell type that can be cultured and cloned from neonatal human skin.…”

Section: Normal Melanocyte Lineagementioning

confidence: 99%

Mechanisms of differentiation in melanoma cells and melanocytes.

Bennett

1989

Environ Health Perspect

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Literature is reviewed on the mechanisms of differentiation in mammalian melanoma cells and normal melanocytes. Pigment cells are particularly useful for studies requiring the observation of differentiation in living cells, for example, studies of commitment. lbpics discussed include melanin synthesis and other markers of pigment cell differentiation; stochastic models of differentiation and commitment; the lability of early stages of differentiation; extracellular factors affecting pigment cell differentiation, with implications for intracellular controls; the role of proliferation and the cell cycle in differentiation, and the relative roles of changes in transcription, translation, and posttranslational processes.

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“…At the time of subculture of melanoma and nevus cultures, cells were split such that slide chambers could be made for immunocytochemical assay and the L-dopa tyrosinase assay [15]. Nevus or melanoma cells were cultured at a density of 2.5 x lo4 cells per well in two chamber/slides (Lab-tek) with 2 ml of F-10 culture medium containing 10% fetal bovine serum.…”

Section: Lmrnunocytochemistrymentioning

confidence: 99%

Melanoma growth stimulatory activity: Isolation from human melanoma tumors and characterization of tissue distribution

Richmond

1988

J of Cellular Biochemistry

148

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Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, auto-stimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio-Gel P30, reverse phase-high performance liquid chromatography and heparin-sepharose steps. This modified procedure provides a 10-fold improved yield of MGSA over previously published procedures. Purified MGSA-stimulated melanoma cell growth in both 3H-thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13-14 Kd based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF beta), and epidermal growth factor (EGF) in combination with TGF beta did not stimulate 3H-thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three-fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.

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False Dopa Reaction in Studies of Mammalian Tyrosinase: Some Characteristics and Precautions

Cited by 17 publications

References 7 publications

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases

Mechanisms of differentiation in melanoma cells and melanocytes.

Melanoma growth stimulatory activity: Isolation from human melanoma tumors and characterization of tissue distribution

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