Detection of Total UDP-Glucuronosyltransferase (UGT) Activity in Melanoma Cells

The UDP-glucuronosyltransferases (UGTs) are integrally involved in the clearance of a wide range of drugs used to combat human diseases. UGTexpression levels and activity can be induced by drug addition to cells and has been proposed as a potential intratumoral drug resistance mechanism. Traditional methods of assaying UGT activity are drug-centric and require HPLCs with multiple detectors (dependent on individ-ual drug). Here, we describe a generalized method to detect total UGTactivity (intrinsic or induced) via the UGT-Glo assay which utilizes a general UGT substrate with luminescence as the readout eliminating the need for multiple HPLC detectors to detect total UGT activity in a given sample. The method detailed here can be applied for any UGT containing sample, allowing for the efﬁcient detection of total UGT activity to be the functional endpoint using a plate reader. In this manner, global changes in UGT activity can be monitored in response to a wide variety of stimuli.


Introduction
The UGT family of enzymes is integrally involved in the detoxification of many carcinogens, the clearance of drugs and the metabolism of a variety of endogenous substrates such as bilirubin, steroid hormones, and bioactive lipids [1,2]. Although UGTs are primarily expressed in liver, they also play vital roles in other tissues in the body. For example, UGT2B15 and UGT2B17 are expressed in the prostate where they regulate local androgen levels through glucuronidation [3] and UGT1A10 and UGT2B7 are expressed in the breast where they regulate estrogens [4]. There is also ample evidence that the UGTs play important roles in the aerodigestive tract, gastrointestinal tract, lung, colon, bladder, kidneys, and brain [5][6][7][8][9]. Recently, UGT2B7, UGT2B10, and UGT2B15 were identified as being normally expressed in human melanocytes [10]. However, UGT expression was observed to be lost during progression of melanocytes to melanoma [10]. Treatment of melanoma cell lines which lack UGT expression with anticancer agents induced expression of UGT2B7, UGT2B10, and UGT2B15 demonstrating that melanoma cells retain the ability to re-express these same three UGTs [10]. The corresponding increase in glucuronidation activity in melanoma cells following anticancer treatment was also observed using the UGT-Glo assay. Since the UGTs are drug metabolism enzymes, this re-expression of the UGTs is proposed to constitute a previously unsuspected mechanism for intratumoral drug resistance in melanoma [10].
Traditional methods of detecting UGT activity involve thin layer chromatography (with a radiolabeled co-substrate) or HPLC [11,12]. In either method, a limiting factor in detecting total UGT activity is in the selection of a general UGT substrate which is a decent substrate for all UGT family members present in the sample. Further limitations can be access to sample, cost, or detection of the substrate. Some substrates have decent UV absorption; others require fluorescence or mass spectrometry detection. Here, we describe a general method to detect total UGT activity in melanoma cells using the UGT-Glo assay. This method has the distinct advantage of using a general UGT substrate that can react with a luciferin detection reagent to produce light that can be detected on a luminometer, unless that substrate is glucuronidated first (Fig. 1). If the substrate is glucuronidated, it will no longer react with the detection reagent and no light will be emitted. Thus, reactions are run in parallel for each reaction, one with co-substrate (UDPGA) and one without co-substrate. In each case the (À) UDPGA reaction is the negative control that will emit light (Fig. 1). The (+) UDPGA reaction is the experimental reaction and any reduction detected in emitted light, as compared to negative control, indicates UGT activity (Fig. 1). These reactions are carried out in a 96well plate (or 384 if desired) and as such several experimental conditions can be assayed at once (even in triplicate) as opposed to injecting one sample at a time onto an HPLC with run times of 15-30 min (or longer) each. However, it is important to note that this technique is not applicable to UGT kinetics assays, studies identifying UGT/substrate specificity or any other substratespecific investigations. In those instances the traditional UGT activity assays are required.

Materials
Prepare all solutions with ultrapure water. Always keep any enzymatically active preparation on ice and minimize the time spent on ice prior to experiment to retain optimum activity.

Tris Buffered Saline (TBS) homogenate buffer: 25 mM Tris
base, 138 mM NaCl and 2.7 mM KCl, pH 7.4. Add 8 g of NaCl, 3 g of Tris base, and 0.2 g of KCl to 800 mL water. Adjust pH to 7.4 and then add water to a final volume of 1 L. Can store at room temperature. To make working solution of TBS homogenate buffer add 10 mL to a clean 15 mL conical and dissolve one pellet of Complete mini protease inhibitor cocktail EDTA-free (Roche). Must be EDTA free as no chelating agents can be present in UGT assay since Mg +2 is required for optimal activity.

Detection of UGT Activity in Melanoma Cells
All cell culture should be carried out using sterile technique in a laminar flow hood (class II).  UDPGA and the (+) UDPGA conditions). To keep total volume equivalent, add water to each sample up to 10 μL total. For a negative control, include a no UGT condition (six wells) in which only 10 μL of water is present. For a positive control, add 1 μL of human liver microsomes (HLM) + 9 μL of water (six wells). Since most UGT family members are highly expressed in HLM, 1 μL is plenty to serve as a positive control. 9. Incubate at room temperature for 20 min to stabilize the luminescent signal.
10. Read luminescence on a plate-reading luminometer or CCD camera. Values will be reported in relative light units (RLU).
3.5 Data Analysis 1. Only the (+) UDPGA wells will have UGT activity and that activity will reduce the amount of RLUs detected. Remember this is due to the fact that if the multienzyme substrate is glucuronidated then it will NOT bind to the D-Cysteine and subsequently react with the Luciferin Detection Reagent (yielding light). Thus, to calculate total UGT activity the RLU value in the (+) UDPGA wells have to be subtracted from the corresponding (À) UDPGA wells for that same experimental condition.
2. Repeat this analysis for each well pair. Since all conditions were performed in triplicate an average and standard deviation can easily be calculated.
3. Final RLUs can then be compared across experimental condition by normalizing to total protein content which was obtained in Section 3.3 above.

Notes
1. SKMel28 metastatic melanoma cells were used in this experiment since they lack UGT expression. Using these cells it is easy to detect which drugs induce UGT activity since any detection of UGT activity would indicate the UGTs had been reactivated. Normal passage conditions for SKMel28 are 1:4 twice a week. Although SKMel28 is used in this example, the procedure is applicable to any cell line to detect intrinsic (or inducible) total UGT activity.
2. Any desired drug, hormone, growth factor, cytokine, carcinogen, etc. can be used here to test the effect on global UGT activity in any desired cell line. We find it is important to treat cells with various concentrations of drug with the lowest concentration being below the IC 50 concentration for the given drug. 7. The UGT multienzyme substrate is used here and is the best option for measuring total UGT activity in a sample. It is important to note however that UGT1A4 has very little activity against this substrate. There is a UGT1A4 specific substrate also included in the UGT-Glo kit and this substrate should be used additionally to the multienzyme substrate if UGT1A4 is known to be present in the homogenate. In our SKMel28 cells UGT1A4 is not present and so only the multienzyme substrate is used.
8. Incubation time for glucuronidation assay can vary. Ninety minutes was plenty for our example, but this time can be extended as desired (even overnight). The caveat is that once the substrate or co-substrate is used up (or UGT enzyme degrades) then the activity will no longer be in the linear range for effective comparisons. Still, if detection of activity is the endpoint then an overnight reaction may be more fitting.
9. The resuspended Luciferin Detection Agent (without D-Cysteine) can be stored at 4 C for 1 week or À20 C for 3 months without loss of activity (per manufacturer instructions).