The firefly luciferase gene is widely used as a reporter gene for studying the regulation of gene expression (1)
, particularly the improved Promega firefly luciferase gene used in mammalian cell expression(2)
. The advantages of the luciferase assay include its sensitivity, and fast and convenient quantitation of gene expression. Normally a second reporter gene (e.g., β-galactosidase) is used as an internal experimental standard to normalize for the differences in transfection efficiencies and other experimental variations. Promega has developed an alternative means for this internal normalization of experimental results, the DLR™ Assay System, a fast and convenient two-reporter assay in a single test tube(3)
Alpha-MSH induces melanin synthesis in cultured melanoma cells(4)
by causing an increase of both tyrosinase enzyme activity (5)
and tyrosinase mRNA synthesis(7)
. Tyrosinase is specifically expressed in melanin-producing cells and melanoma cell lines, as has been shown by the use of a mouse tyrosinase promoter cloned upstream of the chloramphenicol acetyltransferase reporter gene(8)
The influence of some agents on the tyrosinase promoter-luciferase construct has been described(1)
. In this report the authors used the modified luciferase assay for the study of alpha-MSH induction in melanoma cells. The effect of [Nle4, D-Phe7]-α-MSH (NDP-alpha-MSH) on functional activity of human tyrosinase promoter/enhancer region in a newly designed reporter plasmid was monitored by the DLR™ Assay in B16-F1 cells. The tyrosinase promoter is known to be active only in melanocytes and some melanoma cell lines.
To study alpha-MSH specific gene activation, the alpha-MSH inducible plasmid pGL3-Tyr14A containing an improved firefly luciferase gene (luc+)(9)
under the control of human tyrosinase promoter/enhancer region, was evaluated. As a positive control we used the plasmid pGL3-Control Vector (Cat.# E1741), which contains an improved firefly luciferase gene (luc+) under the control of a SV40 promoter/enhancer region. As a negative control we used the pGL3-Basic Vector (Cat.# E1751), containing the improved luc+ gene without a promoter or enhancer. The pRL-SV40 Vector is an expression vector encoding Renilla reniformis luciferase under the control of the SV40 promoter and enhancer. The pGL3-Tyr14A plasmid contains the 2.2kb region of the human tyrosinase promoter/enhancer element cloned upstream from the luc+ gene in the pGL3-Basic Vector and was obtained from Dr. M. Januszeski (Baxter Diagnostics Inc., Irvine, CA).
Cell Culture and Transient Transfections
B16-F1 cells (mouse melanoma cell line CRL-6323; ATCC, Rockville, MD) were maintained in DMEM (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). The cells were cultured at 37°C in a humidified atmosphere (5% CO2). New cultures were initiated from frozen stock every two months. The cells were seeded the day before the transfection at a density of 2 × 105 cells per well in six-well plates (Falcon°, Becton, Dickinson and Co.). The cotransfections with the pGL3-Control, pGL3-Tyr14A or pGL3-Basic Vectors (2µg/well) and the second plasmid pRL-SV40 (0.2µg/well) were performed using SuperFect™ Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The DNA:SuperFect™ ratio was 1:5 (µg:µl) and the cells were incubated with the DNA-SuperFect™ complex for two hours.
Treatment of the B16-F1 Cells with NDP-alpha-MSH
NDP-alpha-MSH (Sigma Chemical Co.) was dissolved in N, N-dimethylformamide (DMF; Sigma Chemical Co.) at a stock concentration of 0.1mM and maintained at –20°C until use. The B16-F1 cells were cotransfected with pGL3-Tyr14A and pRL-SV40, and were divided into two groups: the first group was treated with DMF (control) and the second group with 10–7M NDP-α-MSH. Media with NDP-α-MSH, DMF or without additives was exchanged for fresh media every other day. At the indicated intervals (see legend, Figure 1) all groups were lysed with Passive Lysis Buffer (Cat.# E1941) and assayed for luminescence according to the DLR™ Assay System protocol (#TM040; Figure 1). The light output was measured with a TD-20/20 Luminometer with Dual Auto Injector. The measurements were carried out in triplicate. The firefly luciferase activity was normalized to the Renilla reniformis luciferase activity (Figure 1).
The transient transfection of pGL3-Tyr14A into B16-F1 cells resulted in an enhancement of luciferase activity (4-fold increase) compared to the pGL3-Control Vector. Transient transfection of the promoterless pGL3-Basic Vector resulted in low luciferase activity, barely detectable above background levels. When the pGL3-Tyr14A-transfected B16-F1 cells were exposed continuously to NDP-α-MSH, an increase in luciferase gene expression was observed. The maximum increase of 30% in luciferase activity was observed 72 hours after NDP-α-MSH stimulation (Figure 1).
Previous studies have shown that alpha-MSH increases tyrosinase mRNA levels in melanoma cells(7)
. The amelanotic Cloudman S-91 mouse melanoma cell line showed a maximum increase in tyrosinase mRNA of 6-fold 48 hours after hormone treatment and in enzyme activity of 16- to 20-fold 72 hours after stimulation with alpha-MSH. This data correlates with our data measuring 4-fold increase of the luciferase signal upon stimulation. The hormone-mediated increase in the luciferase levels is consistent with an induction of mRNA synthesis.
The use of the second reporter gene makes it possible to eliminate variability among the samples caused by differing transfection efficiencies or other factors. In 1996 a Dual-Light® Reporter Gene Assay System (Tropix/PE Biosystems) was described, which sequentially detects the firefly luciferase activity and beta-galactosidase luminescent activity with a specific substrate in a single reaction tube(10)
. The disadvantage of this system is that it is time consuming. The Promega DLR™ Assay System uses the Renilla luciferase as an internal control to establish a baseline by which each measurement can be normalized. The use of the DLR™ Assay System with normalization of the two luminescent signals makes it possible to obtain the corrected value from the experiments on induction of the luciferase expression. The advantage of the method described here is the simplicity of the assay. The alpha-MSH-induced stimulation was evaluated by luciferase assay rather than doing much more complex assays for mRNA or enzyme quantitation. The test system described here could be widely used for studies on the influence of selected agents on tyrosinase promoter activity.
We thank Dr. M. Januszeski for plasmid pGL3-Tyr14A and Drs. A. Zilberchtein and V. Medvedkin for their assistance and fruitful discussions.