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In vitro cell based screening

a. Cell-based growth inhibition assays

b. Clonogenic assays

c. Three-dimensional cell cultures
Mechanism Based Screening and the discovery of novel anti-cancer targets
Mechanism of action evaluation
Xenograft and Hollow Fiber Implantation Animal Models
Syngeneic animal models

Anti-Cancer Cell-Based Growth Inhibition Assays
Using a panel of cell lines available through the National Cancer Institute or the American Type Culture Collection, we can evaluate the ability of test molecules to inhibit the growth of representative tumor cells. These cells are standardized based on their doubling time, growth medium requirements and optimal time for endpoint evaluation. Endpoint quantification is performed by a variety of means, including metabolic and macromolecule synthesis assays.

Methodology for evaluating growth inhibition:

	Tetrazolium dye: This methodology measures the ability of mitochondrial enzymes in viable cells to metabolize the tetrazolium dye to a soluble formazan product.
	Tritiated thymidine incorporation: This methodology measures cellular proliferation through incorporation of thyimidine during DNA synthesis.
	Macromolecule synthesis evaluation: (tritiated thymidine for DNA, tritiated uridine for RNA and tritiated leucine for protein): This methodology provides information on the cellular capability to synthesize DNA, RNA and protein.
	Trypan blue dye exclusion: Methodology to allow microscopic quantification of live versus dead cells.
	Colorimetric evaluation with neutral red: Quantification of cellular viability
	Molecular Probes Live/Dead Viability/Cytotoxicity assay: The LIVE/DEAD ™ Viability/Cytotoxicity assay is a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes (calcein AM and ethidium homodimer [EthD-1]) The probes measure two recognized parameters of cell viability: intracellular esterase activity (Calcein AM) and plasma membrane integrity (EthD01).
	Mitochondrial RNA and DNA: Evaluation by PCR to quantify the health of the mitochondria.

Mechanism-Based Screening and the Discovery of Novel Anti-Cancer Targets

I.	Historical Perspective of Anti-Cancer Therapy Chemical agents used as anti-cancer therapeutics today include: nitrogen mustard, methotrexate, chlorambucil, thioguanine, cyclophosphamide, vinblastine, 5-fluorouracil, vincristine, melphalan, daunorubicin, cytosine arabinoside, bleomycin, doxorubicin, mitomycin, dacarbazine, BCNU, cisplatin, etoposide, teniposide, amsacrine, carboplatin, epirubicin, mitoxantrone, paclitaxel, gemcitabine, docetaxel, topotecan, irinotecan Genotoxic (Cytotoxic) Therapy Mitotic poisons DNA-reactive Drugs Inhibitors of DNA replication DNA topology as a target Selectivity Growth Control Pathways The cell cycle clock Stopping and starting the cell cycle clock Programmed cell death The cell cycle calendar Therapeutic strategies Host-Tumor Interactions Low-molecular weight inducers of tumor necrosis Low molecular weight modulators of cytokine effects
II.	Cell Cycle and Cell Cycle Checkpoints Molecular recognition of cell cycle progression: cyclins and cyclin-dependent kinases (Cdks) control the processes of DNA replication and cell division and these controls are deficient in cancer cells Molecular regulation of cell cycle checkpoints: checkpoints are mechanisms that monitor cell regulatory pathways and DNA structure before the cells enter the next phase of the cell cycle; controlled by members of the phosphoinositol kinase (PIK) family; role of p53 and p21 in G1-S checkpoint control; role of Cdc25C in G2-M checkpoint control; checkpoint effector kinases Chk1 and Chk2; role of p53 in G2 checkpoint control Targeting cyclin-dependent kinases and cell cycle checkpoint pathways: oncogenic viruses commonly activate cyclin-dependent kinases and inactivate the checkpoint pathways; the G1-S transition pathways are commonly activated in human cancer; the cell cycle checkpoint pathways are commonly defective in human cancer Agents and strategies for therapeutic interference: Cdk inhibitors; checkpoint kinase inhibitors; other sites for intervention
III.	Growth Factor and Signal Transduction Targets The erbB family of of receptor tyrosine kinases (RTKs) The Ras-Raf-MEK-ERK signaling pathway c-Src kinase, signal transduction, transformation and cancer Akt Nuclear hormone receptors as targets
IV.	Cell Death Pathways Two main pathways for drug-induced apoptosis: the mitochondrial pathway; the death receptor dependent pathway Moldulation of drug-induced cell death by Bcl-2 and related proteins The central role of caspases in drug-induced apoptosis Synergy between death receptors and cytotoxic drugs The Rel/NF-kB/IkB proteins
V.	Drug Resistance Pathways Targeting drug transport Targeting cellular stress responses Targeting DNA repair systems
VI.	Cancer Invasion and Metastasis The extracellular matrix Cancer invasion and metastasis Cell adhesion Cancer cell motility Inflammatory response Proteolytic enzymes implicated in cancer invasion MMPIs as novel anticancer agents Sheddases The uPA system
VII.	Tumor Vasculature Angiogenesis: Fibroblast growth factors; Vascular endothelial growth factor; Angiopoietins Integrins The extracellular matrix
VIII.	Tumor Antigens Antigen targets Tumor antigens = targets for antibody-based therapeutics