Human ovarian carcinoma cell lines, 3AO and AO, are estrogen- and progestogen-dependent. They were obtained from the Cell Bank at the Chinese Academy of Sciences for this study.17-19 Both cell lines were grown in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Evergreen, Hang Zhou, China), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. Exponentially growing 3AO and AO cells (5 × 10⁴ cells/mL) were treated with different concentrations of PROG (Sigma, St. Louis, MO) for 48 hours and 72 hours, respectively.

3AO and AO cell lines were plated in sextuplicate wells of 96-well microtest plates are treated as described.20 At various intervals after PROG was added, the plates were pulsed with 2 μCi ³H-thymidine/well (specific activity, 22 Ci/mmol; Shanghai Institute of Nuclear Sciences, Chinese Academy of Sciences), trypsinized, and harvested on strips of fiberglass filter paper with the use of multiple automated sample harvesters. The radioactivity of individual samples was measured in a liquid scintillation counter.

Drug-treated and untreated 3AO and AO cells (1 × 10⁶) were washed twice with phosphate-buffered saline (PBS) and resuspended in 25 μL PBS. The cells were lysed by the addition of 25 μL lysis buffer (60 mM Tris, pH 7.4; 50 mM ethylene diamine tetraacetic acid; and 1.6% sodium lauryl sarcosine) containing 1 mg/mL proteinase K, incubated for 3 hours at 50 °C, and digested with 200 μg/mL DNAse-free RNAse A for an additional 20 minutes. DNA from the cell lysates was then analyzed on a 1.2% agarose gel containing ethidium bromide, and visualized and photographed under ultraviolet light.21

After being treated with 10 μm PROG and all-trans-retinoic acid (ATRA, Sigma, St. Louis, MO) for 72 hours, cells were centrifuged, and the pellets were gently resuspended in propidium iodide solution (PI; 50 μg/mL in 0.1% sodium citrate plus 0.15 Triton X-100; Sigma, St. Louis, MO).22 Random fields of each treated cell culture were observed under a microscope through a ×40 objective lens in fluorescent mode. Apoptotic cells had condensed nuclei, and the percentage of apoptotic cells was calculated by counting approximately 500 cells.

PROG-treated and untreated 3AO and AO cells (2 × 10⁶) were washed twice with PBS containing 0.1% glucose and then fixed in 1 mL ice-cold ethanol overnight at 4 °C. The fixed cells were pelleted and resuspended in 0.5 mL of PBS containing 0.1% glucose, 30 μg/mL PI, and 1 mg/mL RNAse A (Sigma, St. Louis, MO). The DNA contents of the cell were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA) as described.23

Cell cycle distribution was determined by DNA content, as assayed by propidium iodide staining. The percentage of cells in each phase of the cell cycle was determined with the Cellfit software provided by Becton-Dickinson (San Jose, CA) as described.24

Extraction of total RNA and Northern blot analysis were performed as described.25 The human DNA probes used in this study were from the Pst I cDNA insert of pMG-WAF1 plasmid for wild-type p53,26 the EcoRI/Hind III cDNA insert of plasmid pFL1 for bcl-2,27 and the EcoRI cDNA insert of plasmid pGDSV7 for c-myc.28 Thirty μg of total RNA were loaded in each lane of a 1% agarose gel containing 3% formaldehyde and transferred to nylon membranes. Blots were hybridized to the probes radiolabelled to specific activity of 1-2 × 10⁹ cpm/μg with α-³²P dATP (Amersham, Buckinghamshire, United Kingdom). Then the blots were exposed to X-ray films (Kodak, New York, NY) for 3-5 days. The membranes were rehybridized with a β-actin cDNA probe to serve as an internal control.

Cell proliferation was dramatically inhibited by PROG treatment in both ovarian carcinoma cell lines (Fig. 1). After 72 hours, 1 μM PROG inhibited ³H-thymidine incorporation by 23% and 21% and 10 μM PROG by 69% and 73% in 3AO and AO cell lines, respectively. In contrast, the inhibitions after 48 hours of treatment with both concentrations of PROG were less significant than those after 72 hours of treatment. Therefore, PROG treatment at 10 μM for 72 hours was chosen for the subsequent experiments.

After 72 hours of 10 μM PROG treatment, as shown in Figure 2, agarose gel electrophoresis of DNA from the apoptotic cells showed the characteristic DNA fragmentation ladder. Under the same conditions, the percentage of apoptotic cells reached 70 ± 9% in 3AO cells and 49 ± 7% in AO cells, as measured by the counting method (Fig. 3). Under the same conditions, cell apoptosis was approximately 76 ± 6% in 3AO cells and 55 ± 6% in AO cells, as determined by the sub-G1 peak in the flow cytometry histograms (Fig. 4). The two independent assays in this study gave similar results, and thus clearly demonstrated that PROG could indeed promote apoptosis in the ovarian carcinoma cells tested. The ability of PROG to induce apoptosis is apparently higher than that of ATRA (also at 10 μM for 72 hours), which is reported to induce apoptosis of breast carcinoma cells.29

The nuclear DNA content of individual cells in each population was determined by flow cytometry. The results are presented in Figure 5. Seventy-two hours after treatment with PROG, the peak representing 3AO cells with G₁ DNA content had increased from 37.0 to 72.5%, and from 53.5 to 73.8% in AO cells, whereas the fraction of cells in S-phase had decreased from 31.9 to 11.3% in 3AO cells and from 26.6 to 13.4% in AO cells.

Among the apoptosis-related genes we tested, after the cells were treated with 10 μM PROG for 72 hours, only the level of p53 mRNA markedly increased in both 3AO and AO cells as detected by Northern blot analysis (Fig. 6). The mRNA levels of bcl-2 and c-myc, however, were not significantly changed by the PROG treatment in the two cell lines (data not shown).