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The following are assays of cancer chemotherapeutic and chemopreventive agents that are commonly used in Dr. You’s lab:

A. Cell growth assays: We have routinely used a series of human lung cancer cell lines and a series of mouse lung tumor cell lines. For chemoprevention experiments, we also use immortalized cells and primary culture of normal lung epithelial cells. Cell growth is determined in six- or 24-well culture plates. Cells are seeded in their respective culture media at densities that varied as a function of their proliferation rate. Cells are allowed to grow for 2 days and then treated for 5-6 days with different concentrations of putative cancer therapeutic or chemopreventive agents. Treatments for lung cancer cell lines are performed in 0.1% FBS, and 1% FBS. Media are renewed every 2 days. At the end of treatment, cells are harvested and the cell number is determined using a cell counter. Cell proliferation is also evaluated by a colorimetric method based on the cellular conversion of tetrazolium salt (MTT) into a blue formazan product.

B. In vivo studies using nude mice. Athymic nude mice (4-6-week-old) are obtained from the Jackson Laboratory (Bar Harbor, ME). The 100,000 to 1,000,000 cells in log phase growth will be injected subcutaneously into each of the two flanks of the athymic nude mice. Either one-week prior to injections of tumor cells or at the time of the injections of tumors, mice will be given either i.p. injection or orally (i.g.) for the duration of the experiment. The health of the animals and the size of tumors will be monitored daily. Tumor latency (day) and the tumor size (mm3) will be recorded. The experiment was terminated at four to six weeks after the injection. After each mouse is terminated, tumors from the primary sites will be removed from mice and the weight (gram) will be carefully measured. In addition, gross examination will be conducted to assess for the presence of smaller tumors and possible metastases. Portion of the tumor will be excised and fixed in 10% buffered formalin for histopathological evaluations.

C. In vivo lung tumor models: A/J mice and transgenic mice. Dr. You’s lab has used both A/J mice and various transgenic mice for chemotherapeutic and chemopreventive efficacy of putative agents. A/J mice are highly susceptible to lung tumor induction with chemical carcinogens and the transgenic mice contain K-ras activating mutations, deletion of wild type K-ras allele, p53^val135 dominant-negative mutation, and deficient for p16. The protocol for the treatment of A/J and transgenic mice with putative agents is as follows. Six-week-old female A/J mice or mutant mice will be randomized into control groups and treatment groups. Animals in control groups will be administered a single i.p. dose of a carcinogen in 0.1 ml saline and additional treatment with vehicles for the putative agents. In treatment groups, animals will be treated with putative agents (one-week before, two weeks after, or 12 weeks after carcinogen treatment) and will continue to 20 weeks. All the animals from each group will be terminated by CO₂ asphyxiation 20 weeks after carcinogen treatment. The portion of lung adenocarcinomas will be removed and flash frozen in liquid nitrogen for molecular analysis. The remaining lung will be fixed in 10% buffered formalin for histopathological analysis. For each lung, the number (N), volume (V), and total lung tumor volume (N x V) will be measured. Student’s t-tests or ANOVA will be run to test for significance response of the tumors to the treatments.

D. Cell cycle studies. If growth suppression activity of a given agent is found, we usually conduct further assays for the ability of the test agent to induce a cell cycle arrest or increase in apoptosis in cells described in In Vitro assays (Part A). Briefly, the baseline cell cycle distribution of lung cancer cell lines will be determined by Flow Cytometry using a standard propidium iodide (PI) DNA labeling technique. We will also compare the cell cycle distribution under normal and low serum conditions. Serum deprivation should not trigger cell cycle arrest in the parental lung tumor cell lines treated with an agent or with the vehicle. Analysis of DNA content is performed essentially as reported previously. Cells are grown under normal or selective conditions (serum content), and harvested for analysis. Cell nuclei are subjected to propidium iodide, which stains double-stranded DNA. The samples are read on an Epics Elite Flow Cytometer (Coulter) which utilizes an air-cooled 15 mw argon laser operating at 488 nm. The histograms are analyzed by the M-Cycle program (Coulter). This technique is reproducible, giving a percentage of the total population in each phase of the cell division cycle (sub-G0/G1; G0/G1; S; G2/M).

E. Apoptosis studies. For assaying apoptosis, we will evaluate the level of apoptosis in vitro comparing the control lung cancer cell lines and the cells treated with a given agent by Flow Cytometry. The PI technique for cell cycle can be used to indicate the percent of the total population in a sub-G1 state (apoptotic cells). In addition, we will conduct a more specific assay for apoptosis. The TUNEL assay (Terminal Uridine Nucleotide End Labeling) exploits a product of apoptosis, namely the 3'-Hydroxyl ends produced by the enzymatic digestion of the DNA during apoptosis. These break sites are a target for the enzyme terminal deoxynucleotidyl transferase (TdT). Using a modification of the standard TUNEL assay, the TdT enzyme then incorporates bromodeoxyuridine triphosphate (Br-dUTP) at the break site. The apoptotic events are then scored using a fluorescein-tagged anti-BrdU monoclonal antibody for detection. Apoptotic events for Flow Cytometry will be assessed using the Apo-BrdU Fluorescein kit (Pharmingen). Cells are grown under normal or selective conditions (serum content), and harvested for analysis. The procedures are essentially as stated previously, and follow the manufacturer's guidelines. The nuclei are exposed to the TdT enzyme, which incorporates a molecule of Br-dUTP at the 3'OH sites produced during apoptosis. The samples are then incubated with a fluorescein-labelled anti-BrdU monoclonal antibody, followed by staining total DNA with PI, as before. The data will be collected as before on an Epics Elite Flow Cytometer (Coulter), measuring the red fluorescence of PI, and now the green fluorescence of fluorescein, to generate a log FL1 vs. linear FL2 dot plot. The negatively stained cells should fall within the first log decade on the Y axis (green). PI intensity is read on the X-axis (red), and differentiates between debris, sub-G1, G0/G1, S, and G2/M phase cells.

F. Biomarker and mechanistic studies. These studies are various and unique to each test agent.