Cell culture
SKLMS1, SKUT1, and SKMEL2 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and are listed in Additional file 1. WT cancer cell lines were grown in MEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Bio-Techne, Minneapolis, MN), 1.3% 100× penicillin-streptomycin (10,000 U/mL) (Thermo Fisher Scientific), and 2.5 μg/mL Plasmocin (InvivoGen, San Diego, CA). LTAT cells were grown in this medium with 1 μg/mL ADI-PEG20 (Polaris, San Diego, CA) added. All ADI-PEG20 treatments were performed by replacing media with media that had been pre-treated with 1 μg/mL ADI-PEG20 for at least 8 h at 37 °C.
Mouse embryonic fibroblasts (MEFs) were generated as detailed later. MEFs were grown in IMDM (Thermo Fisher Scientific) supplemented with 20% FBS, 1% 100× MEM non-essential amino acids (Thermo Fisher Scientific), 0.0007% 2-mercaptoethanol (MilliporeSigma, Burlington, MA), 1% 100× penicillin-streptomycin (10,000 U/mL), and 2.5 μg/mL Plasmocin.
Automated cell imaging
Cells were transduced with IncuCyte® NucLight Red Lentivirus Reagent (Essen BioScience, Ann Arbor, MI) and selected with puromycin. SKLMS1, SKUT1, and SKMEL2 cells were plated in 96-well plates at 3 × 103, 7.5 × 103, and 5 × 103 cells per well, respectively. The next day, fresh phenol red-free medium was added to start treatment along with 50 nM YOYO™-1 Iodide (Thermo Fisher Scientific), which fluorescence green within the nuclei of dead cells. Images were taken with IncuCyte® ZOOM or IncuCyte® S3 (Essen BioScience) automated fluorescent microscopes at a rate of 0.5–1 per hour. Red and green cell numbers were quantified automatically with an analysis algorithm within the IncuCyte software (Essen BioScience).
SKLMS1, SKUT1, and SKMEL2 cells expressing the indicated GFP arginine sensor variants were plated in 96-well plates at 3 × 103, 7.5 × × 103, and 5 × 103 cells per well, respectively. The next day, fresh phenol red-free medium was added to start treatment. Glutamine deprivation experiments used media supplemented with One Shot™ dialyzed FBS (Thermo Fisher Scientific) in place of standard FBS, and 2 mM L-glutamine (Corning, Corning, NY) was either added or omitted. Images were taken with IncuCyte® ZOOM or IncuCyte® S3 automated fluorescent microscopes at a rate of 0.5–1 per hour. Sensor fluorescence was quantified automatically with an analysis algorithm within the IncuCyte software, and average integrated intensities of green fluorescence in individual cells were taken.
SILAC RPMI 1640 Flex Media, with no glucose and no phenol red (Thermo Fisher Scientific) was used in experiments to vary arginine concentrations. Glucose (Agilent Technologies, Santa Clara, CA), l-glutamine, and l-lysine hydrochloride (MilliporeSigma) were added to the same concentrations as RPMI 1640 (Thermo Fisher Scientific), along with 10% dialyzed FBS and 1.3% pen/strep. l-arginine (MilliporeSigma) was added to the indicated concentrations. Cells were grown and passaged in RPMI 1640 for at least 1 week before arginine concentration experiments. Fluorescence of the arginine sensor was measured with the IncuCyte S3 at 0, 1, and 2 h of the indicated treatments. The areas under the curve for this time period were then plotted against arginine concentration, with 0 represented as 0.01 μM. Data were fit to the following equation:
$$ Y=\mathrm{Bottom}+\frac{\left({X}^{HillSlope}\right)\times \left( Top- Bottom\right)}{\left({X}^{HillSlope}+ EC5{0}^{HillSlope}\right)} $$
Effective concentration (EC) of any other value (F) was then calculated with the following equation:
$$ \mathrm{ECF}={\left(\frac{F}{100-F}\right)}^{\sqrt{HillSlope}}\times EC50 $$
where indicated, 100 μM cycloheximide (CHX) (MilliporeSigma) or 1 μM bortezomib (BTZ) (Millennium Pharmaceuticals, Cambridge, MA) was added to media. Fluorescence data in the presence of CHX were fit to the following exponential one-phase decay equation:
$$ Y=\left({Y}_0- Plateau\right)\ast {e}^{-K\ast X}+ Plateau $$
Half-lives were calculated as (ln(2)/K).
Individual cell tracking
Arginine sensor fluorescence of individual cells was tracked over time by hand within the IncuCyte S3 software by recording the integrated intensity of green objects (nuclei) detected by the analysis algorithm. One hundred cells were initially targeted for tracking in each experimental condition. Results include data from less than 100 cells, as not all cells were able to be tracked with certainty. At timepoints where fluorescence in a cell was too low to be detected by the algorithm, the integrated intensity value was recorded as 0.
Capillary electrophoresis
For obtaining lysates after a time course of ADI-PEG20, SKLMS1 cells were plated in 60-mm dishes, and treatments were administered so that all ended simultaneously. 7.5 × 104 LTAT cells were plated for every timepoint. 7.5 × 104 WT cells were plated for samples with 12 or fewer hours of treatment, and the number was doubled for each full day of treatment, with 6 × 105 cells plated for 72 h. Treatment was started by the addition of fresh media the next day. All samples received fresh media 2 h before harvesting. After lysis, immunoblots were performed with a Wes automated immunoblot machine (Bio-Techne) according to the manufacturer’s protocol [25]. Each sample was normalized to its own total protein [25]. Two ASS1 primary antibodies were used: a non-commercial mouse monoclonal from Polaris and a rabbit polyclonal ab175607 (Abcam, Cambridge, MA). Mouse monoclonal GFP antibody sc-9996 (Santa Cruz Biotechnology, Dallas, TX) was used to detect the arginine sensor. Two bands were detected at approximately the expected molecular size of the GFP sensor, and both were quantified and added together before normalization to total protein. Although a single band was expected, the identity of the two separate bands was not investigated. Antibodies are listed in Additional file 2.
Cloning
A gBlocks® Gene Fragment (Integrated DNA Technologies, Coralville, IA) of the first arginine sensor variant (ArgSen (-) NLS), containing a P2A site and Fast-FT without a nuclear localization sequence (NLS), was cloned using AscI and NotI restriction sites. All gBlocks® in this study were ordered with extra nucleotides on each end to allow for direct restriction digestion and ligation into the vector. All arginine sensor variations were first cloned into plasmid pKLV2-EF1a-BsdCas9-W (replacing the original insert), then subcloned into plasmid pLV-EF1a-IRES-Puro. pKLV2-EF1a-BsdCas9-W was a gift from Kosuke Yusa (Addgene plasmid # 67978 ; http://n2t.net/addgene:67978 ; RRID:Addgene_67978) [26]. pLV-EF1a-IRES-Puro was a gift from Tobias Meyer (Addgene plasmid # 85132; http://n2t.net/addgene:85132; RRID:Addgene_85132) [27]. Successful cloning was confirmed by Sanger sequencing with primers EF1a fwd, pKLV2 seq rev, pLV seq rev, and Rad23b end fwd. All oligonucleotides in this study were ordered from Integrated DNA Technologies and are listed in Additional file 3.
After cloning ArgSen (-) NLS, multiple modifications were made. A C-terminal SV40 Large T-antigen NLS was added by amplification of Fast-FT with SalI BamHI FastFT fwd and NotI NLS FastFT rev primers, followed by replacement of Fast-FT with Fast-FT-NLS using BamHI and NotI restriction sites (ArgSen). Separately, the P2A site was removed by SalI restriction digestion followed by ligation (Argsen (-)P2A (-)NLS). Fast-FT in ArgSen (-)P2A (-)NLS was then replaced by enhanced green fluorescent protein with a C-terminal NLS (GFP-NLS) that had been PCR amplified with SalI BamHI GFP fwd and NotI NLS GFP rev primers, using BamHI and NotI restriction sites (GFP ArgSen). The polyarginine region in this variant was then replaced with a region of identical length encoding random non-arginine amino acids, obtained as a gBlock® and cloned with restriction sites XbaI and EcoRI (GFP RanSen).
Variations of the sensor with regions deleted to test degradation were derived from GFP ArgSen. DNA oligonucleotide pairs designed to replace targeted regions with short linkers were phosphorylated on their 5’ ends by T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA), then annealed. After annealing, each double-stranded oligonucleotide possessed complementary overhangs with the correct sequences to bind to the digested restriction sites on either side of its target region. The target region was cut out of the vector at these sites, and the newly annealed oligonucleotide was ligated in. NucGFP was made with Reporter only oligonucleotides using AscI and BamHI restriction sites. Δ Degradation Domain was made with Proteasomal del oligonucleotides using AscI and BstBI restriction sites. Δ Disordered Region was made with Disordered del oligonucleotides using AscI and NsiI restriction sites. Δ Rad23b UbL was made with Rad23b del oligonucleotides using NsiI and XbaI restriction sites. After publication, all constructs will be available from Addgene (Watertown, MA).
Lentiviral particles were made with Lenti-X™ 293 T cells (Takara Bio USA, Mountain View, CA). Experimental cells were transduced with virus and selected with puromycin.
Flow cytometry
SKLMS1 ArgSen cells were plated in 6-well plates for a time course of ADI-PEG20, along with control WT cells for each timepoint. To enable simultaneous harvesting at similar levels of confluency, the following numbers of cells were plated per well according to the length of ADI-PEG20 treatment: 1 × 105 for 72 h, 6.5 × 104 for 48 h, 4 × 104 for 24 and 18 h, and 2.5 × 104 for 12 or fewer hours. All samples received fresh media 2 h before harvesting. Cells were washed with PBS, harvested with trypsin, and measured for fluorescence of blue Fast-FT by flow cytometry with a 407-nm laser and 50-nm wide bandpass filter centered at 450 nm. Fluorescence values of paired WT samples were subtracted from ArgSen samples, and values were then normalized to untreated fluorescence levels.
RT-qPCR
For isolating RNA after a time course of ADI-PEG20 treatment, SKLMS1 cells were plated in 60-mm dishes, and treatments were administered so that all ended simultaneously. 7.5 × 104 cells were plated for samples with 12 or fewer hours of treatment, and double for each full day of treatment, with 6 × 105 cells plated for 72 h. All samples received fresh media 2 h before harvesting. Cells were washed in ice-cold PBS, scraped, and centrifuged. RNA was isolated by Direct-zol™ RNA Miniprep Plus kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to produce cDNA according to the manufacturer’s protocol. The Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) was then used to quantify arginine sensor and GAPDH mRNA according to the manufacturer’s protocol. qrtPCR ArgSen fwd, qrtPCR ArgSen rev, qrtPCR GAPDH fwd, and qrtPCR GAPDH rev primers were used in the RT-qPCR reactions.
Protein translation assay
SKLMS1 WT cells were plated at 6 × 103 cells per well in a 96-well plate. The next day, media was replaced and indicated treatments were given at indicated times so that the assay began simultaneously for all samples. The Click-&-Go Plus 647 OPP Protein Synthesis Assay Kit (Click Chemistry Tools, Scottsdale, AZ) was used according to manufacturer’s instructions. Red fluorescence was measured on the IncuCyte S3, and cell-by-cell analysis was performed to quantify the mean intensity of fluorescence in each cell. The average fluorescences of CHX controls for each timepoint were subtracted from each sample before normalization to the untreated 0 timepoint.
Mouse embryonic fibroblast generation
HEPD0731_5_F08 mutant embryonic stem cell clones were ordered from the European Conditional Mouse Mutagenesis Program (EUCOMM). These cells contain an allele of ASS1 harboring FRT-flanked lacZ and neomycin resistance genes, followed by a loxP site upstream of critical exon 4 and another loxP site downstream of exon 4. Embryos were grown from these stem cells, and the resulting mice were crossed with mice expressing FLP recombinase. This cross removed the cassette containing lacZ and neomycin resistance genes, allowing the ASS1 allele to encode functional ASS1 protein. These mice were then backcrossed to C57BL/6 mice with confirmed FLP-negative genotypes three times to remove FLP and possess ASS1F/+ alleles. ASS1F/+ mice were crossed to each other, and a colony of ASS1F/F mice was established.
MEFs were generated from embryos harvested from an ASS1F/F × ASS1F/F mating. MEFs were allowed to grow normally and became spontaneously immortalized. MEFs were infected with either Ad5CMVCre or Ad5CMVcytoLacZ adenoviral particles (University of Iowa, Iowa City, IA) to knock out ASS1 or serve as a negative control, respectively. Complete knockout of ASS1 was confirmed by both genotyping and immunoblot.
Metabolomics
For metabolomic analysis, SKLMS1 WT and LTAT cells expressing GFP ArgSen were plated in replicates of four on 100 mm dishes at 2 × 106 cells per dish for every condition except LTAT with 48 h of ADI-PEG20, which had 1 × 106 cells. The next day, cells were treated with ADI-PEG20 for the indicated time periods, with each sample getting fresh media 1 h before harvesting. Viable cells were counted for one sample from each condition, to be used for normalization. Metabolites were extracted from the remaining three samples with methanol using the protocol for extraction of metabolites from adherent cells from Human Metabolome Technologies America (HMT) (Boston, MA). Purified metabolites and viable cell counts were then sent to HMT for metabolomic analysis. Data from HMT were then normalized to ratios of individual sample total metabolites to average total metabolite levels per 1 × 106 cells. The concentrations of arginine in ADI-PEG20-treated MEM and untreated MEM were also determined by HMT.
Cell volume determination
For determining cell volume after 48 h of ADI-PEG20 treatment, cells were plated and treated identically to the metabolomics experiment, then harvested by trypsin. Samples were mixed well, and 20 μL of each were taken, mixed with an equal volume of trypan blue (Thermo Fisher Scientific), pipetted into Countess™ Cell Counting Chamber Slides (Thermo Fisher Scientific), and analyzed by a Countess™ II Automated Cell Counter (Thermo Fisher Scientific). Average cell diameter was calculated from this data. The remaining cells were analyzed by flow cytometry, measuring forward scatter intensity to determine relative cell diameters. Individual and average cell volumes were then calculated with FlowJo software (FlowJo, Ashland, OR).
Statistics
Data were analyzed using GraphPad Prism 8 Software (GraphPad Software, San Diego, CA). mRNA and GFP immunoblot error bars represent standard error of the mean. All other error bars represent standard deviation. ASS1 protein expression differences were analyzed by unpaired t test. Growth rate differences were analyzed by one-way ANOVA. Metabolite differences at 48 h were analyzed by Welch’s t test. “ns” denotes non-significant difference between groups. * denotes p < .05, ** denotes p < .01, *** denotes p < .001, and **** denotes p < .0001.