Tissue Culture drugs
Upon optimization of ex vivo maintenance, cell culture soon became the model of choice to test and discover drug function and efficacy. Today, an extensive repertoire of compounds are available to scientists to achieve the effect of choice because of cell culture-based functional characterizations. Below is a brief introduction to TC-drug experimental design, followed by a summary of compounds organized by function.
TC drug treatments: Experimental design
Upon initial use of a compound in cell culture, two factors must be optimized: treatment dose and treatment time. Often, the literature may narrow a range of conditions that are likely to contain the optimal dose and time, but ultimately, these must be empirically determined and verified. In order to identify these conditions, one of these variables is often fixed, while another is tested. Often, the drug to assay is not received in a solubilized form. To begin this assay, solubilize the drug in a vehicle (often an organic solvent) to create a master stock as outlined below.
Organic solvent vehicles
To minimize introduction of organic solvent vehicle into tissue culture, make a 100 mM concentrated stock of drug in DMSO and then dilute in aqueous solutions, such as PBS or culture medium, for intermediate dilutions up to 10 mM. Store stocks at -80C in appropriate aliquot amounts that prevent the need for repeated freeze thaw. Upon retrieval from -80C, thaw and store on ice until ready to add to to culture cells/tissue. Ensure that the vehicle volume never exceeds 0.1% of the culture media volume (i.e. 1:1000 or 1μl/ml).
For dose-response assays, determine max concentration to be used in the dose response, and ensure that the frozen working stock is at least 1000x more concentrated. For example:
TSA max dose = 10μM. Therefore TSA stock concentration = 10mM.
5-aza max dose = 10μM. Therefore 5-aza stock concentration = 10mM.
Upon initial use of a compound in cell culture, two factors must be optimized: treatment dose and treatment time. Often, the literature may narrow a range of conditions that are likely to contain the optimal dose and time, but ultimately, these must be empirically determined and verified. In order to identify these conditions, one of these variables is often fixed, while another is tested. Often, the drug to assay is not received in a solubilized form. To begin this assay, solubilize the drug in a vehicle (often an organic solvent) to create a master stock as outlined below.
Organic solvent vehicles
To minimize introduction of organic solvent vehicle into tissue culture, make a 100 mM concentrated stock of drug in DMSO and then dilute in aqueous solutions, such as PBS or culture medium, for intermediate dilutions up to 10 mM. Store stocks at -80C in appropriate aliquot amounts that prevent the need for repeated freeze thaw. Upon retrieval from -80C, thaw and store on ice until ready to add to to culture cells/tissue. Ensure that the vehicle volume never exceeds 0.1% of the culture media volume (i.e. 1:1000 or 1μl/ml).
For dose-response assays, determine max concentration to be used in the dose response, and ensure that the frozen working stock is at least 1000x more concentrated. For example:
TSA max dose = 10μM. Therefore TSA stock concentration = 10mM.
5-aza max dose = 10μM. Therefore 5-aza stock concentration = 10mM.
List of TC Drugs by Category
- HDAC inhibitors - Inhibit HDACs to establish acetylated chromatin in euchromatic state
- Mitotic Inhibitors - inhibit mitosis by disrupting microtubule assembly
- DNA Methylation inhibitors
- Polymerase inhibitors - inhibit polymerases to cease DNA or RNA synthesis. Ex. nucleoside analogs, Doxorubicin (topoisomerase), ActinomycinD (RNA pol),
- Ribosome inhibitors - inhibit ribosomal translation of RNA to protein. Ex. cyclohexamide
- DNA Damage via UV
- Actinomycines - antitumor antibiotic - ActinomycinD - Blocks RNA pol progression on DNA template.
Mitotic Inhibitors
1) Taxanes: stabilize GDP-bound tubulin in the microtubule to halt the process of cell division in a 'frozen mitosis'
- Examples: Paclitaxel, Doxetaxel (chemotherapeutics)
2) Vinca Alkaloids: Inhibit microtubule polymerization to destroy mitotic spindle
- Examples: Vinblastine, Vincristine, Vindesine, Vinorelbine (chemotherapeutics)
3) Colchicine: binds tubulin to inhibit microtubule polymerization (not chemotherapeutic, only TC, sometimes for gout)
Nucleoside analogs
1) Thymadine analogs (not a nucleoside)
- Bromodeoxyuridine (BrdU): substitutes a Br group for thymidine's CH3 group. Used to measure cell proliferation using BrdU-specific antibodies
De-methylating Drugs
HDAC Inhibitors: Establish euchromatic genome
“classical” HDIs act on Class I and Class II HDACs by binding to the zinc-containing catalytic domain of the HDACs. Grouped below in order of decreasing potency:
The sirtuin Class III HDACs are dependent on NAD+ and are, therefore, inhibited by nicotinamide, as well derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.[14]
Additional functions
HDIs should not be considered to act solely as enzyme inhibitors of HDACs. A large variety of nonhistone transcription factors and transcriptional co-regulators are known to be modified by acetylation. HDIs can alter the degree of acetylation nonhistone effector molecules and, therefore, increase or repress the transcription of genes by this mechanism. Examples include: ACTR, cMyb, E2F1, EKLF, FEN 1, GATA, HNF-4, HSP90, Ku70, NF-κB, PCNA, p53, RB, Runx, SF1 Sp3, STAT, TFIIE, TCF, YY1, etc.[11]
Ultraviolet
Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible light but longer than X-rays (from 400 nm to 10 nm in wavelength).
UVB light can cause direct DNA damage.
UVA - 400 – 315 nm
UVB - 315 – 280 nm
UVC - 280 – 100 nm
actinomycines
Actinomycin binds to a premelted DNA conformation present within the transcriptional initiation complex. This immobilizes the complex, interfering with the elongation of growing RNA chains by RNA polymerase. This drug is used in cell biology to inhibit active transcription and assess mRNA stability of already existing transcripts.
Ribosome inhibitors - inhibit ribosomal translation of RNA to protein.
Cycloheximide (CHX) inhibits de novo eukaryote protein synthesis at the translational elongation step by blocking tRNA translocation at the ribosome. Common experimental usage includes treating cells with cycloheximide in a time-course experiment followed by Western blotting of the cell lysates for the protein of interest to assess differences in protein half-life without confounding contributions from transcription or translation.
MG132 - proteosome inhibitor
MG132 is a specific, potent, reversible, and cell-permeable proteasome inhibitor used to inhibit degradation of ubiquitin-conjugated proteins in mammalian cells to block protein turnover.
CDK inhibitors
PD0332991 - CDK4/6 inhibitor
NU6140 - CDK2 inhibitor
R03306 - CDK1 inhibitor
- HDAC inhibitors - Inhibit HDACs to establish acetylated chromatin in euchromatic state
- Mitotic Inhibitors - inhibit mitosis by disrupting microtubule assembly
- DNA Methylation inhibitors
- Polymerase inhibitors - inhibit polymerases to cease DNA or RNA synthesis. Ex. nucleoside analogs, Doxorubicin (topoisomerase), ActinomycinD (RNA pol),
- Ribosome inhibitors - inhibit ribosomal translation of RNA to protein. Ex. cyclohexamide
- DNA Damage via UV
- Actinomycines - antitumor antibiotic - ActinomycinD - Blocks RNA pol progression on DNA template.
Mitotic Inhibitors
1) Taxanes: stabilize GDP-bound tubulin in the microtubule to halt the process of cell division in a 'frozen mitosis'
- Examples: Paclitaxel, Doxetaxel (chemotherapeutics)
2) Vinca Alkaloids: Inhibit microtubule polymerization to destroy mitotic spindle
- Examples: Vinblastine, Vincristine, Vindesine, Vinorelbine (chemotherapeutics)
3) Colchicine: binds tubulin to inhibit microtubule polymerization (not chemotherapeutic, only TC, sometimes for gout)
Nucleoside analogs
1) Thymadine analogs (not a nucleoside)
- Bromodeoxyuridine (BrdU): substitutes a Br group for thymidine's CH3 group. Used to measure cell proliferation using BrdU-specific antibodies
De-methylating Drugs
- 5-azacytidine: De-methylating drug
HDAC Inhibitors: Establish euchromatic genome
“classical” HDIs act on Class I and Class II HDACs by binding to the zinc-containing catalytic domain of the HDACs. Grouped below in order of decreasing potency:
- hydroxamic acids (or hydroxamates), such as trichostatin A,
- cyclic tetrapeptides (such as trapoxin B), and the depsipeptides,
- benzamides,
- electrophilic ketones, and
- the aliphatic acid compounds such as phenylbutyrate and valproic acid.
The sirtuin Class III HDACs are dependent on NAD+ and are, therefore, inhibited by nicotinamide, as well derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.[14]
Additional functions
HDIs should not be considered to act solely as enzyme inhibitors of HDACs. A large variety of nonhistone transcription factors and transcriptional co-regulators are known to be modified by acetylation. HDIs can alter the degree of acetylation nonhistone effector molecules and, therefore, increase or repress the transcription of genes by this mechanism. Examples include: ACTR, cMyb, E2F1, EKLF, FEN 1, GATA, HNF-4, HSP90, Ku70, NF-κB, PCNA, p53, RB, Runx, SF1 Sp3, STAT, TFIIE, TCF, YY1, etc.[11]
- Trichostatin A (TSA): HDAC inhibitor of class I and II HDACS (TSA inhibits HDACs 1, 3, 4, 6 and 10 with ) with IC50 values around 20 nM. Results in euchromatic genome with altered gene expression. TSA inhibits the eukaryotic cell cycle during the beginning of the growth stage by altering the ability of transcription factors to access the DNA.
Ultraviolet
Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible light but longer than X-rays (from 400 nm to 10 nm in wavelength).
UVB light can cause direct DNA damage.
UVA - 400 – 315 nm
UVB - 315 – 280 nm
UVC - 280 – 100 nm
actinomycines
Actinomycin binds to a premelted DNA conformation present within the transcriptional initiation complex. This immobilizes the complex, interfering with the elongation of growing RNA chains by RNA polymerase. This drug is used in cell biology to inhibit active transcription and assess mRNA stability of already existing transcripts.
Ribosome inhibitors - inhibit ribosomal translation of RNA to protein.
Cycloheximide (CHX) inhibits de novo eukaryote protein synthesis at the translational elongation step by blocking tRNA translocation at the ribosome. Common experimental usage includes treating cells with cycloheximide in a time-course experiment followed by Western blotting of the cell lysates for the protein of interest to assess differences in protein half-life without confounding contributions from transcription or translation.
MG132 - proteosome inhibitor
MG132 is a specific, potent, reversible, and cell-permeable proteasome inhibitor used to inhibit degradation of ubiquitin-conjugated proteins in mammalian cells to block protein turnover.
CDK inhibitors
PD0332991 - CDK4/6 inhibitor
NU6140 - CDK2 inhibitor
R03306 - CDK1 inhibitor