Cancer Prevention and Intervention Core Laboratory

Laboratory Manager: David Yu, Ph.D.

Cancer is the result of abnormal genetic control of cell growth that arises from structural changes in DNA or mutations. Many mutations occur in somatic cells during the lifetime of an individual and some may result in cancer. There is evidence that dietary factors can impede this process. For example, epidemiological studies reveal that the quarter of the population with the lowest dietary intake of fruit and vegetables has roughly twice the cancer risk for most types of cancer compared with the quarter with the highest intake. In recent years the possible chemoprevention of cancer by specific chemical constituents of fruit and vegetables has attracted much attention.

Established in April 2002, the Cancer Prevention and Intervention (CPI) Core Laboratory at the Linus Pauling Institute primarily provides genetic toxicology testing service to our researchers. The mission of this laboratory is to help investigators learn whether the dietary compound of interest is a potential chemoprotective agent and to elucidate the possible inhibitory mechanism(s). We employ several techniques, including the Salmonella mutagenicity assay (or Ames test), single cell gel electrophoresis assay (SCG or Comet assay), and micronucleus (MN) assay.

These assays enable investigators to assess the mutagenic, DNA-damaging and clastogenic effects of chemicals. Using compounds identified as mutagens or clastogens as "positive controls," we are able to study the potential beneficial activities of dietary compounds or any chemical entity of interest against those DNA-damaging agents.

Individuals or organizations outside of the OSU campus may also order these testing services. Please contact David Yu (541-737-5082 or [email protected]) for further information.


Photo of automatic plate readerThe Salmonella Mutagenicity Assay

The Salmonella mutagenicity test, or bacterial reverse mutation assay, is also commonly known as the Ames test. It was developed by Dr. Bruce Ames and his colleagues in UC Berkeley. The principle of the test is to expose histidine-dependent Salmonella typhimurium strains (the tester strains, which have artificially induced point mutations) to a compound to be examined in a histidine (His) deficient medium. His-independent bacterial colonies may arise from spontaneous reversions (backward mutations) or chemically induced reversions. The mutagenicity of a chemical can be assessed by comparing the control with the treated bacterial culture. Conversely, the antimutagenicity of a compound to a selected positive mutagen can be investigated when the two chemicals are co-administered to the bacteria.

 


Photo of imaging systemThe Comet Assay

The comet assay, or single cell gel electrophoresis assay, is used to detect DNA strand breaks as well as alkaline labile lesions. For many labs, it has become the tool of choice for detecting DNA damage in eukaryotic cells due to its versatility. Exposed and control cells are embedded in an agarose gel sandwich on a microscope slide. The cells are then subjected to electrophoresis. When the electrophoresis is performed in a neutral pH condition, DNA double strand breaks are revealed. In strong alkaline solution, all DNA strand breaks and alkaline labile lesions are revealed. The migration of DNA is visualized under a fluorescence microscope after the DNA is stained with a fluorescent dye. The shape of DNA distribution is very similar to a celestial comet. Measurement is usually carried out on a computerized image analysis system, like the one shown in the picture at right from the CPI Core Lab. In principle, all types of nucleated cells may be used in this assay.

 


The Micronucleus Assay

The micronucleus (MN) assay can be used to detect the clastogenic and aneugenic effects of test chemicals both in vitro and in vivo. It is much more cost effective than the metaphase chromosome aberration assay. The in vitro micronucleus assay is considered as the replacement for conventional metaphase analysis as the screening test of choice for clastogenicity. The in vivo assay, usually conducted in mice, is especially important since no in vitro alternative test has been validated to replace the MN test. The cells evaluated in this assay are typically erythrocyte populations in either the peripheral blood or bone marrow compartment. Clastogens or spindle poisons that cause chromosomal damage in stem cells result in the formation of readily detectable micronuclei in the anucleated immature erythrocytes.

 


Histone Deacetylase (HDAC) Inhibition Assay

Epigenetic changes are thought to play an important role in carcinogenesis; for example, the methylation of DNA can affect the interaction of proteins (e.g. transcription factors) with DNA, thus affecting gene transcription. Histone acetylation is another type of epigenetic modification that is able to regulate gene expression. In general, inhibition of cellular histone deacetylase activity may lead to loosened histone-DNA binding, resulting in more permissive chromatin DNA to transcription factors. HDAC modulation can lead to transcriptional activation or silencing of a subset of genes controlling cell-cycle arrest, apoptosis, and differentiation. This makes HDAC an interesting and novel target for cancer chemoprotection. The CPI Core Lab provides access to HDAC inhibition assays, for evaluating the chemoprotective potentials of dietary components.

 


Guava PCA-based Assays

Guava Personal Cell Analyzer

Studies of cell cycle kinetics and expression of cell surface/intracellular markers are important in cancer research. The CPI Core Lab acquired a Guava Personal Cell Analyzer (PCA), a compact and accessible platform to several cell analysis assays. The PCA provides forward scatter (cell size) and two fluorescent channels (580 and 675 nm) with a minimal sample requirement. The PCA can be used for:

  • cell count and viability determination
  • apoptosis (TUNEL, nexin, multi-caspases, etc.)
  • cell cycle
  • quantitation of cell surface/intracellular markers.

 

 

The Comet Assay

The Comet AssayLPI/CPI protocol for the Comet Assay, Alkaline Version

1. Equipment and non-chemical materials
1.1 Electrophoresis power supply: Thermo EC 1000-90 or alternative
1.2 Electrophoresis tank: Large Fisher Scientific Recirculating Horizontal System or alternative
1.3 Frosted slides: Surgipath or alternative
1.4 Coverglasses: Surgipath 24 x 50 mm or alternative
1.5 Microcentrifuge and microcentrifuge tubes
1.6 Various micro-pipet and pipet tips
1.7 Disposable test tubes
1.8 Dishes for lysis of cells: Wheaton 900170
1.9 Nikon E400 Fluorescence microscope or equivalent
1.10 Refrigerator and other general laboratory items, e.g. balance and pH meter
 
2. Chemicals and reagents
2.1 10 x concentrated Dulbecco's PBS, Mg2+ and Ca2+ free
    NaCl (Sigma S7653) 80 g
    KCl (Fisher Scientific P217-500) 2 g
    Na2HPO4.2H2O (Fisher Scientific S472-500) 11.5 g
    KH2PO4 (Sigma P0662) 2 g
  Adjust pH to 7.2 and q.s. to 1 liter with dd H2O, autoclave store at RT.
2.2 Dulbecco's PBS, Mg2+ and Ca2+ free
  Dilute 10 times of solution 3.1 with dd H2O, final pH 7.3 ~ 7.4. Autoclave at 121°C for 20 min, store at RT.
2.3 Lysing solution for the comet assay (equivalent to 2 liters in the final preparation with the addition of 1% Triton X-100 and 10% DMSO)
  2.4 g Tris (Sigma T6066), 74.4 g Na2EDTA.2H2O (Sigma E5134), 292.2 g NaCl (Sigma S7653), appropriate amount of H2O.
  Set pH to 10 with NaOH pellets (Sigma S0899), q.s. to 1780 ml with dd H2O, store at RT.
2.4 10 N NaOH (Sigma S0899) stock solution (Caustic!!! Handle with care)
  200 g q. s. to 500 ml with dd H2O, store at RT.
2.5 200 mM EDTA (Sigma E5134) stock solution
  Dissolve 74.4 g in dd H2O, set pH to 10 with NaOH, q. s. to 1000 ml with dd H2O, store at RT.
2.6 0.5% low melting point agarose (LMPA)
0.5 g + 100 ml PBS(A), boil in microwave oven to dissolve agarose, compensate the evaporated H2O, aliquot the agarose into 15 ml tubes (~3 ml/ tube) before gelation and store the tubes under 4°C.
2.7 1.0% normal melting point agarose (NMPA)
  1 g + 100 ml dd H2O, microwave to dissolve agarose, compensate the evaporated H2O, cool down to around 60°C before use.
2.8 Neutralizing buffer - 0.4 M Tris (Sigma T6066)
  Dissolve 48.5 g in dd H2O, set pH 7.4 with HCl and q. s. to 1000 ml with dd H2O, store at RT.
2.9 Electrophoresis solution – 300 mM NaOH, 1 mM EDTA
  Mix 48 ml of 10 N NaOH, 8 ml of 200 mM EDTA and 1544 ml dd H2O. Chill to 4°C before use.
2.10 Fluorescent staining solution – 250 x Strength Stock
Ethidium bromide (Sigma E8751, DNA poison!!! Handle with care) in dd H2O 5 mg/ml, store at RT.
2.11 Staining solution
  Dilute solution 3.10 250 time to 20 µg/ml with dd H2O, store at RT.
2.12 Dimethyl sulfoxide (Fisher Scientific D128-500)
2.13 Triton X-100 (Sigma T9284)
 
3. Operation procedures
3.1 Material and reagent preparation
3.1.1 Mix 89 ml lysing solution (3.3) with 1 ml of Triton X-100 and 10 ml of DMSO in a staining dish, chill the mix to 4°C before use.
3.1.2 Microwave the LMPA and NMPA aliquots to complete molten state (in a container filled with water), check and shake the tube every a few seconds to avoid overheat and explosion. Leave the molten LMPA in 40°C bath and NMPA in 45°C for at least 10 min before use.
3.2 Slide preparation
Note: Steps 3.2.1 may be carried out days before Comet assay experiment.
3.2.1 Dip ¾ of clean frosted slides (unfrosted portion), one at a time, in molten NMPA. Scrape agarose off the underside. Leave the slides horizontally to dry. A slide warmer may be used to facilitate the drying of slides.
Note: Conduct the steps 3.2.2 to 3.2.3 under dimmed light to prevent the occurrence of DNA damage.
3.2.2 Mix about 10,000 cells with 75 µl of molten 0.5% LMA at 37°C. Pipette the cell suspension onto the first agarose layer, and spread the mixture with a coverglass, and maintain the slide on an ice-cold flat tray for 30 min.
3.2.3 After removal of the coverglass, add the third layer of 0.5% LMA (70 µl) at 37°C, spread gel using a coverglass and again allow the gel to solidify on ice for 10 min.
3.2.4 After removal of the coverglass, immerse the slide in freshly prepared cold lysing solution for a minimum of 1 h at 4°C.
3.3 Electrophoresis
3.3.1 Remove the slides from the lysing solution, drain the slides and rinse briefly with cold dd H2O. Place in a horizontal gel electrophoresis tank side by side, avoiding spaces and nearest to the anode. Fill the electrophoresis tank with 1.6 L of cold electrophoresis solution.
3.3.2 Leave the slides in the solution for 30 min to allow the unwinding of the DNA and expression of alkali-labile damage.
3.3.3 Perform electrophoresis at 4°C for 30 min (or other desirable time) at a constant voltage (0.8 V/cm).
3.4 Staining
3.4.1 After electrophoresis, Place the slides horizontally and gently add Tris buffer (0.4 M Tris, pH 7.5) dropwise to neutralize the excess alkali. Let the slides sit for 5 min. This neutralizing procedure was repeated three times.
3.4.2 Add 60 µl EtBr (20 µg/ml) to each slide and cover it with a coverglass. Let the slides sit for 5 min at the horizontal position. Place the slides in a humidified airtight container to prevent drying of the gel. Analyze the slides within 3-4 h.
3.5 Slide scoring
Randomly score the comet-like nuclei using the Comet Assay Image Analysis System. For each treatment score a minimum of 50 cells or 25 cells per slide is required.

The Micronucleus Assay

The micronucleus assayLPI/CCP protocol for in vitro micronucleus assay using CHO-K1 cells

1. Equipment and materials
1.1 Tissue culture vessels (e.g. flasks or plates)
1.2 CHO-K1 cell line (from ATCC)
1.3 Rat liver S9 (Aroclor-1254 induced), if metabolic activation system is to be used
1.4 9” disposable Pasteur pipets
1.5 Frosted slides (Surgipath #00240 or alternative)
1.6 Coverglasses (Surgipath 24 x 50 mm, #00145, or alternative)
1.7 General purpose bench-top centrifuge (Fisher Scientific Marathon 3000)
1.8 Conical centrifuge tubes (Falcon, VWR #21008-918)
1.9 Wheaton stainless steel 30-slide racks and glass staining dishes
1.10 Various micro-pipets and pipet tips
1.11 Nikon E400 fluorescence microscope or equivalent
1.12 Other general laboratory items, e.g. freezer, balance and pH meter
 
2. Chemicals, reagents and media
2.1 Ham's F12K medium (Mediatech, through Fisher Scientific) supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Fisher Scientific MT25035CI), 10% fetal bovine serum (FBS)
2.2 Ham's F12K medium supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate
2.3 Trypsin-EDTA solution, (Fisher Scientific ICN1689649)
2.4 Cytochalasin B (VWR IC19511910,), 3 mg/ml in DMSO, sterile
2.5 Dulbecco's PBS (A), Mg2+ and Ca2+ free (may be prepared from 10 x stock solution)
  Ingredient per 1000 ml
  NaCl (Sigma S7653) 8.0 g    
    KCl (Fisher Scientific P217-500) 0.2 g    
    Na2HPO4 (Fisher Scientific S472-500) 1.15 g    
    KH2PO4 (Sigma P0662) 0.2 g    
  Adjust pH to 7.3 ~ 7.4 and q.s. to 1 liter with dd H2O. Autoclave at 121°C for 20 min. Keep sterile and store at RT.
2.6 Fixative, made fresh
  Ingredient per 26 ml  
    Methanol (Fisher Scientific A452-4) 25 ml    
    Acetic acid, glacial (Fisher Scientific A35-500) 1 ml    
2.7 10 µg/ml acridine orange (VWR # EM-1125) in PBS (A), good for 1 week on bench
2.8 Potassium/magnesium solution (for S9 mix)
  Ingredient per 500 ml  
    KCl (Fisher Scientific P217-500) 61.5 g    
    MgCl2 6H2O (Fisher Scientific, BP214-500) 40.7 g    
    dd H2O to 500 ml    
  Dissolve the salts. Autoclave at 121°C for 20 min. When the solution has cooled slighted, tighten cap. Keep sterile and store at RT.
2.9 0.2 M Sodium phosphate buffer, pH 7.4 (for S9 mix)
    Ingredient per 500 ml  
    NaH2PO4 (Fisher Scientific BP329-1) 12.0 g    
    Na2HPO4.2H2O (Fisher Scientific, S472-500) 17.8 g    
    dd H2O to 500 ml    
  Dissolve the salts with 450 ml dd H2O. Adjust pH with 0.2 M NaH2PO4 or 0.2 M NaHPO4 to 7.4. Add dd H2O to the final volume of 500 ml.
Autoclave at 121°C for 20 min. When the solution has cooled slighted, tighten cap. Keep sterile and store at RT or 4°C.
2.10 80 mM NADP (VWR# 80053-340, for S9 mix)
  Ingredient per 1 ml
    NADP (F.W. 765.39) ~61.23 mg  
    Sterile dd H2O ~1 ml*  
  It is better to weigh a batch of dry aliquots of NADP in small sterile tubes with tight caps. Avoid light and moisture. Store them at -20ºC or below. Add dd H2O according to the exact weight of NADP upon use. This is an expensive reagent – make up solutions as required and not in excess.
2.11 120 mM D-Glucose-6-phosphate (G-6-P, VWR ICN100312, for S9 mix)
  Ingredient per 1 ml    
    G-6-P (F.W. 304) ~36.48 mg    
    Sterile dd H2O ~1 ml*    
  Prepare as for NADP above.
2.12 Fresh High S-9 mix with cofactors
    Ingredient per 10 ml final concentration    
    Sterile dd H2O 2.8 ml      
    0.2 M phosphate buffer, pH 7.4 5.0 ml 0.1 M    
    80 mM NADP 0.5 ml 4 mM    
    120 mM G-6-P 0.5 ml 6 mM    
    K, Mg salts solution 0.2 ml 33 mM – 8 mM    
    Rat liver S-9 (Aroclor-1254 induced) 1.0 ml 10% v/v    
  Actual volume should be determined by each experiment. Make up solutions as required and not in excess. Prepare immediately before use in ice bucket in above order. Keep the ingredients chilled in preparation. Add in S9 fraction last. Discard any leftover for S9 or S9 mix. Never re-use S9 or S9 mix.
   
3. Operation procedures
3.1 Culture CHO-K1 cells in ATCC recommended growth medium in exponentially proliferating status. Prepare a cell suspension at a density ~1 × 106 cells /ml. Seed cells in culture vessels at 3 × 104 cells /0.3 ml (cell suspension : medium = 1 : 9)/cm2. The number of subcultures is determined by the following formula:
(# of dose levels) x (# of treatment regimen, i.e. 4 h/20 h, +S9/-S9) x (3 replicates)
3.2 Grow the subcultures until they reach 60 ~ 70% confluence.
3.3 Prepare the treatment medium by adding the test compound to a desirable amount of growth medium (including 3 µg/ml cytochalasin B, for 20 hr treatment) or serum free medium (for 4 hr treatment). Use metabolic activation system (rat liver S9) for 4 h treatment when necessary. Normally negative and positive controls should be included.
3.4 Remove the medium in the culture vessels by aspiration and immediately add the same amount of treatment medium in the vessels. Do not expose the cells to air for more than a few seconds.
3.5 Skip this step for 20 hr treatment experiment. For 4 h treatment, remove the treatment medium by aspiration. Wash the cells with a same amount of serum free medium once. Remove the serum free medium and add fresh growth medium, including 3 µg/ml cytochalasin B. Culture the cells for another 20 h.
3.6 Remove the growth medium from cultures by aspiration. Wash the monolayer twice with the same amount of PBS (A). Remove PBS (A) and add 40 µl/cm2 of Trypsin-EDTA to the culture vessel. Incubate and check from time to time the cells. When all the cells are detached, add 0.3 ml/cm2 of growth medium to stop trypsinization.
3.7 Pellet the cells at 250 x g, 5 min in conical centrifuge tubes. Aspirate most of the medium, only leave about 0.5 ml.
3.8 Resuspend the cells in single cell suspension by vigorous pipeting or vortex mixing. Set vortex mixer to a low/intermediate speed. Place a cell suspension-containing tube on the top and slowly add 5 ml of cool fresh fixative. Leave the tubes on bench for 15 min.
3.9 Pellet the cells at 250 x g, 5 min. Decant the fixative to an organic waste bottle. Add fresh fixative (~100,000 cells/ml) to the tube.
3.10 Resuspend the cells using a 9” Pasteur pipette or using intermediate vortex mixing. Drop 8-10 drops of the cell suspension on a horizontally held slide. For each independent treatment, at least 3 slides should be made. Leave the slides on a bench and air-dry the slides. Check the cell density with a phase contrast microscope (40x Ph objective, condenser at Ph2).
3.11 If cells are not to be scored immediately, the dried slides may be stored in N2 filled zip bag in –20°C until use.
3.12 After thoroughly air dry, stain the slides with 10 µg/ml acridine orange for 2 min. Rinse the slides with dd H2O for 4 min. Immediately cover the wet slides with coverglasses. Keep the cells on slide moisturized during the scoring. Score the slides under a 20x objective. Use higher magnification to examine the details.
3.13 Following micronucleus scoring criterion, score 1000 binucleated cells per independent culture.