Chapter 2 Materials and Methods

Cell Culture

For Density Dependent Studies:

Clone 8 C3H-10T½ murine embryonic fibroblasts and clone 16 MCA-10T½ methylcholanthrene-transformed fibroblasts were obtained from the American Type Culture Collection (ATCC). Methylcholanthrene-transformed fibroblasts were derived from treating clone 8 C3H fibroblasts with 3-methylcholanthrene for 24 hours.

Fibroblasts were seeded at 680, 1000, or 5000 cells per milliliter per 2.0cm² growth area (24-well dishes, Falcon) in Basal Media Eagle (BME, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 40 mg/ml gentamicin (Gibco). Cells were incubated at 37°C, 5% CO2 in 95% air. Growth medium was replaced on days 4 and 7. To determine cell number, individual wells were trypsinized in 0.025% trypsin-phosphate buffered saline (Gibco) and resuspended in isotonic buffered saline for particle-size counting (Coulter).

For Biochemical Studies (Growth Factor/Inhibitor Treatment):

Murine embryonic fibroblasts (C3H-10T½, ATCC) were seeded at 1000 cells/ml in Costar culture trays in Basal Media Eagle (BME, Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 40 mg/ml gentamicin (Gibco). Cells were incubated at 37°C, 5% CO2 in 95% air. Growth medium was replaced on days 4 and 7. Confluent cells were staged (serum deprived) on day 10 in Basal Media Eagle supplemented with 0.5 % FBS for 24 hours before pre-incubation with rapamycin ("RP", Calbiochem) or wortmannin ("WT", Calbiochem) for 15-30 minutes. Cells were stimulated with 10 ng/ml TGF-β1 (Bristol-Myers Squibb).

Calculation of Generation Time

To derive generation time, change in cell number was assigned as parameter Y and change in time was assigned as parameter X. Based on laws of natural exponential function (Stewart, 1987), the equations for the parameter Y as a function of X may be expressed as:

Y = exp X where Y intercepted at 1

If X was a rational number, exp X may be defined as a function of e:

e^x = exp X, where e was defined such that its natural log (ln) was 1:

ln e = 1

When the Y-intercept occurred as a function of a rate constant k, e may be expressed as a function of k:

e^kx = exp X, where Y intercepted at a function of rate constant k. Based on the above definitions, Y may be expressed thus:

Y (k) = exp X (k) = e^kx

For generation time, N_f was the cell number observed at the final log phase window; Ni was the initial cell density at the initial log phase window; Tg was the generation time; T was the designated log phase window. In these calculations, T was observed to be from 48-177 hours.

Y = DN = (N_f - N_i)

X = DT = (T/T_g)

Thus the change in cell number relative to change in generation time was:

Y = 2^X

ln Y = X ln 2

ln Y = 0.693 X

ln N_f - ln N_i = 0.693 (T/T_g)

ln (N_f/N_i) = 0.693T/T_g

T_g = 0.693T / ln (N_f/N_i)

Preparation of Radiolabeling Solution

Ten milliliters of BME staging (low-serum) media contained 0.02 ml ³H-Thymidine (Thd, ICN) and 0.05 ml of a 3 mM unlabeled thymidine (Sigma) solution. Unlabeled thymidine was at 500-fold excess to radiolabeled thymidine and was added to achieve Km of the cellular nucleic acid transporter. Cells in each 2.0 cm² well received 0.05 ml of the 10-ml radiolabeling solution. The total well volume including drug and radiolabel addition was 0.6 mls.

For system A amino acid transport assays, ten milliliters of phosphate buffered saline (PBS, Gibco) contained 20 mM of ¹⁴C-methyl-aminoisobutyrate (¹⁴C-MeAIB) and 10 mM MeAIB (500-fold excess to radiolabeled MeAIB, Sigma). Each well received 0.05 ml of the 10-ml radiolabeling solution. The final well volume at time of labeling was 0.55 mls. Total radiolabeling time was 5 minutes.

Radiolabeling Assay

Tritiated substrate was added at time of growth factor treatment unless otherwise specified. Upon completion of labeling, cells were washed with cold phosphate-buffered saline (PBS, Gibco) and acid-soluble fractions were extracted with 2% trichloroacetic acid (TCA, Sigma) to obtain substrate uptake data. TCA-treated cells were rinsed with PBS and air-dried thoroughly before extracting with 0.2N of sodium hydroxide (NaOH, Fisher Scientific) for 4 hours at room temperature to obtain substrate incorporation data. Solubilized aliquots per well were counted in 20 milliliters of scintillation counting fluid (Ecoscint). Radiolabeling assay data were expressed as quantity of substrate (as decays-per-minute or as nanomole) per cell number or cell volume.

System A Amino Acid Transport Measurement

For MeAIB-uptake assays, cells were cultured to confluence and staged overnight before stimulating with increasing concentrations of TGF-β for 24 hours. 25 nanomolar of growth inhibitor was added for an additional incubation of 24 hours before cells were washed with warm PBS. Intracellular amino acid pools were depleted for ten minutes with modified Hank's Balanced Salt Solution supplemented with 1 g/L MgSO₄, 0.2 g/L CaCl₂, and 0.0977 g/L glucose (HBSS from Gibco, reagents from Sigma). Cells were labeled for five minutes with 0.05 mCi/well of ¹⁴C-MeAIB. Labeled cells were washed with ice-cold PBS for subsequent 2% TCA extraction and counted via liquid-scintillation counting. System A transport data were expressed as nanomoles of MeAIB per cell number or cell volume.

Isolation of Whole Cell Extract

Cells were scraped into ice-cold phosphate-buffered saline (PBS, Gibco) and pelleted. Cells were then solubilized in lysis buffer [Per 500 mls PBS: 1% Triton-X100, 10 mM b-glycerophosphate, 0.1% Tween-20, 1 mg/ml aprotinin, 1 mg/ml leupeptin] on ice for 10 minutes to obtain whole cell extracts. Total protein concentration of each extract obtained was determined via Pierce BCA-assay according to vendor's instructions. Extracts were aliquotted and stored at -100°C for up to 6 months.

Immunoblot Analysis

Equal amounts of whole cell extracts (50-100 mg total protein/well) were loaded into 4 - 20% gradient mini-gels (Novex) for SDS-PAGE analysis. Gels with resolved proteins were equilibrated for 15 minutes in transfer buffer [14.4 g glycine, 3.03 g Tris-Base, 20% methanol per liter of HPLC-grade water] before electrophoretic transfer onto PVDF membranes (Novex). Acrylamide gels were stained with Pierce Gel-Code blue according to vendors instructions. Membrane-captured proteins were blocked overnight at 4°C in Pierce Superblock-PBS. Blocked membranes were incubated with antibodies (p27kip1 , p34CyclinD1 , p34Cdk4, p34Cdk2, or p35eIF-2α polyclonal antibodies from Santa Cruz Biotech, p70 S6K^T389 or p70 S6K^T421/S424 polyclonal antibodies from New England Biolabs). Membranes were washed 3X15 minutes in PBS-Tween-20 (0.1%) before incubation with HRP-conjugated secondary antibodies (Amersham) for 1 hour. Membranes were visualized via enhanced chemiluminescence (ECL, Amersham) on photographic film (Kodak X-Omat).