Suicide gene-dependent apoptosis induction was tested in vitro in transduced 293T, MSCs, and GBM cells. In detail, for all cell lines, 5000 cells were seeded in the wells of a 96-well black plate and the following day, Ganciclovir (GCV) (Sigma Aldrich) and Rapamycin (MedChem Express) were added to the TK-transduced cells or RC9-transduced cells respectively at different concentrations (1, 5, 10, 50, 100 ug/ml for GCV and 0.25, 1, 5, 25, 100 nM for Rapamycin). After 24 h (for RC9 cells) or 48 h (for TK cells) of incubation, the luminescence emission after incubation with coelenterazine (Caliper Life Sciences) was measured with GloMax-Multi Microplate Reader (Promega Corporation) and cell viability in each condition was estimated as the luminescence signal ratio with respect to untreated control. In case of DS transduced cells, both assays were performed as described and viability was detected by incubating the cells with CellTiter-Glo 2.0 Cell Viability Assay (Promega) before measuring luminescence emission. All experiments were performed two times in technical triplicate.
Ganciclovir gcv
Manufactured by Merck Group
Sourced in United States
Ganciclovir (GCV) is a synthetic antiviral compound used in the treatment of cytomegalovirus (CMV) infections. It is a nucleoside analogue that inhibits viral DNA synthesis, preventing the replication of the CMV virus.
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Lab products found in correlation
Mg132, by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions)
Normocin, by InvivoGen (2 mentions)
Thrombopoietin tpo, by Thermo Fisher Scientific (2 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions)
Dmem cell culture medium, by Corning (2 mentions)
β mercaptoethanol β me, by Merck Group (2 mentions)
X vivo 15 serum free hematopoietic cell medium, by Lonza (2 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Flt3 ligand, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
16 protocols using ganciclovir gcv
1
Suicide Gene-Dependent Apoptosis Induction
2
Compound Screening for Biological Assays
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The following compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the indicated concentrations were used throughout the study if not specifically mentioned: ARP101 (10 μM), ganciclovir (GCV; 5 μM), MG132 (10 μM), bafilomycin A1 (50 nM), K67 (25 μM), and cycloheximide (CHX; 100 μg/mL). Torin-1 (250 nM), TBCA (tetrabromocinnamic acid; 30 μM), and CK1-7 (50 μM) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA).
3
Cytotoxicity Evaluation of Cisplatin and Ganciclovir
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5 × 103 cells were seeded in the 96-well plate overnight; the next day, diluted cisplatin (Fresenius Kabi, Kemoplat, Solan, India) or ganciclovir (GCV; Sigma, G2536, MO, USA) was added to the medium and incubated at 37 °C, 5% CO2 for 72 h. After incubation, 250 μg of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, SI-5655) was added into each well for 3 h and measured by the plate reader (TECAN, Infinity® 200 PRO, Männedorf, Switzerland).
4
Cell Culture and Prodrug Preparation
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HEK-293, HeLa, and A431 (ATCC) cells were cultured in DMEM supplemented with stable glutamine, sodium pyruvate, 10% fetal calf serum, 1% penicillin/streptomycin (v/v), 4.5 g/l glucose (1.5 g/l for MCF7 cells) to a maximum confluence of 80% and then split 1:10 (HEK-293, MCF7, A431), or 1:50 (HeLa), respectively. All cell culture media and chemicals where purchased from PAA.
Prodrug solutions containing Ganciclovir (GCV, Sigma) or 5-fluorocytosine (5-FC, Sigma) were prepared as ten-fold concentrates (2 mM and 5 mM) in PBS followed by sterile filtration.
Prodrug solutions containing Ganciclovir (GCV, Sigma) or 5-fluorocytosine (5-FC, Sigma) were prepared as ten-fold concentrates (2 mM and 5 mM) in PBS followed by sterile filtration.
5
Preparation of Cell Culture Media
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α-Galactosylceramide (αGC, KRN7000) was purchased from Avanti Polar Lipids. Recombinant human IL-2, IL-3, IL-4, IL-7, IL-15, Flt3-Ligand, Stem Cell Factor (SCF), Thrombopoietin (TPO), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) were purchased from Peprotech. Ganciclovir (GCV) was purchased from Sigma.
X-VIVO 15 Serum-free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including Penicillin-Streptomycine-Glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and Sodium Pyruvate, were purchased from GIBCO. Beta-Mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), Sodium Pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/ml). Medium for culturing human MM.1S tumor cell line (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Adherent cell culture medium (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
X-VIVO 15 Serum-free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including Penicillin-Streptomycine-Glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and Sodium Pyruvate, were purchased from GIBCO. Beta-Mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), Sodium Pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/ml). Medium for culturing human MM.1S tumor cell line (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Adherent cell culture medium (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
6
Modulation of Cellular Pathways
7
Genistein-Mediated Sensitization of Cancer Cells
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9L and M21 cells were seeded in 48 well-plates for 48 hours until they reach 70–80% confluence. Then, cells were transduced with RGD4C-AAVP or control non-targeted vector carrying thymidine kinase of the herpes simplex virus type 1 (RGD-HSVtk) gene with or without 2 hours pretreatment with genistein (150 μΜ). Ganciclovir (GCV, Sigma) was added to the cells (20 μM) at day 3 post vector transduction and renewed daily. Cancer cell killing was quantified at 0, 24, 48, 72, 96 hours post GCV treatment. Cells were counted by using the trypan blue exclusion methodology. Results were normalized to non-targeted vector (fd-HSVtk).
8
Synthesis and Characterization of CDK7 Inhibitors
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The synthesis and antiviral characterization of the parental, selective CDK7 inhibitor LDC4297 (Lead Discovery Center GmbH, Dortmund, Germany) have previously been described in detail [43 (link),44 (link),45 (link),47 (link),52 (link),53 (link),54 (link)]. The covalently binding derivatives QRS6, QRS7, and QRS were designed and synthesized by Qurient Co., Ltd. (C-dong, Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of Korea; Figure S1 ). The antiherpesviral drugs ganciclovir (GCV; Sigma-Aldrich, St. Louis, MO, USA), cidofovir (CDV; Pharmacia & Upjohn S.A., Luxembourg, Luxembourg), and maribavir (MBV; MedChemExpress, Monmouth Junction, NJ, USA) were purchased from the indicated sources and used as reference compounds. Stock aliquots were prepared in DMSO (Sigma-Aldrich, USA) at 10 mM and stored at −20 °C.
9
Generation of Immune Cell Cultures
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α-Galactosylceramide (αGC, KRN7000) was purchased from Avanti Polar Lipids. Recombinant human IL-2, IL-3, IL-4, IL-7, IL-15, Flt3-Ligand, Stem Cell Factor (SCF), Thrombopoietin (TPO), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) were purchased from Peprotech. Ganciclovir (GCV) was purchased from Sigma.
X-VIVO 15 Serum-Free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including penicillin-streptomycin-glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and sodium pyruvate, were purchased from GIBCO. beta-mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), sodium pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/mL). Medium for culturing human Raji and HL60 tumor cell lines (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Medium for culturing HEK 293T cell line (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
X-VIVO 15 Serum-Free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Corning Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including penicillin-streptomycin-glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and sodium pyruvate, were purchased from GIBCO. beta-mercaptoethanol (β-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), sodium pyruvate (1 mM), β-ME (50 mM), and Normocin (100 mg/mL). Medium for culturing human Raji and HL60 tumor cell lines (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Medium for culturing HEK 293T cell line (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
10
MCMV Infection and Ganciclovir Treatment in BALB/c Mice
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5-week-old female BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). For MCMV infection, mice were intraperitoneally (i.p.) inoculated with 1 × 106 pfu of MCMV. At 3, 7, 14, 70, and 90 days post infection (dpi), animals were sacrificed and tissues removed for subsequent analysis [34 (link)]. For ganciclovir studies, mice were treated by i.p. administration of 50 mg/kg/day ganciclovir (GCV) (Sigma Aldrich, Saint Louis, MO, USA) dissolved in PBS. Ganciclovir treatment began 1 day prior to infection and was administered twice daily for 14 days [36 (link)]. Mice were maintained under specific pathogen-free conditions at Louisiana State University School of Veterinary Medicine, Baton Rouge, LA, USA. All animal protocols were approved by the Institutional Animal Care and Use Committee at Louisiana State University.
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