Huh7 cells, a human hepatoma cell line, were maintained in Modified Eagle's medium (MEM) supplemented with 10% heat inactivated fetal bovine serum and an antibiotic/antifungal solution. Cell cultures were maintained at 37℃ under 5% CO₂. All cell culture materials were purchased from Gibco BRL (Gaithersburg, MD, USA).

Albumin in the conditioned medium released from Huh7 cells was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). To observe the dose response of CsA (Sandoz, Seoul, Korea) to albumin synthesis, the cells were plated in six-well dishes, grown to confluence, and incubated in 1 ml of fresh serum-free MEM medium with or without various doses of CsA. After 24 hours, the conditioned medium was collected, centrifuged at 10,000 × g (4℃) for 3 minutes, and stored at -70℃ until assay. For the albumin ELISA, rabbit anti-human albumin antibody (1:4,000) (DAKO, Glostrup, Denmark) was used as the capture antibody and horseradish peroxidase-conjugated rabbit anti-human albumin antibody (1:2,000) (DAKO) was used as the detection antibody. O-Phenylenediamine dihydrochloride (Gibco BRL) was used as the enzyme substrate, and human serum albumin (Green Cross Corp., Seoul, Korea) was used as the standard.

Confluent Huh7 cells, plated in six-well dishes, were incubated in 1 mL of fresh serum-free medium with or without various doses of CsA for 24 hours, and the medium was collected. The conditioned medium was centrifuged at 16,000 × g for 15 minutes at 4℃, and protein concentration of the conditioned medium was determined using the Bradford protein bioassay (Bio-Rad, Hercules, CA, USA). The conditioned medium was heated at 94℃ for 5 minutes in sample buffer. Proteins were separated by SDS-PAGE on 7.5% polyacrylamide gels and were electroblotted onto a Bio-Blot nitrocellulose membrane (Costar, Cambridge, MA, USA). Nonspecific binding was blocked by incubating the blots for 1 hour in 5% (w/v) nonfat milk. Albumin was detected by an overnight incubation with a rabbit anti-human albumin antibody (1:1,000). The primary antibody incubation was followed by six washes in Tris-buffered saline with 0.005% Tween20. The blot was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Life Science, Little Chalfont, UK) at 1:2,000 for 30 minutes. Antibody reactive protein was detected using enhanced chemiluminescence (Amersham Life Science).

Conf luent Huh7 cells, plated in 10 cm dishes, were inc ubated in 4 ml of fresh serum-free medium with or without various doses of CsA for 24 hours to test the effect of CsA on albumin gene expression. For the time course, cells were incubated with or without CsA (10^-6 M) for the indicated time points. RNA was isolated by one-step guanidium-thiocyanate-phenol-chloroform extraction. RNA concentration was measured by absorbance at 260 nm. RNA quantification and integrity were confirmed by 1% agarose gel electrophoresis and ethidium bromide staining. Total RNA was fractionated by electrophoresis on a 1% agarose-formaldehyde gel and blotted by capillary transfer onto a nylon membrane (Gene Screen, NEN, Boston, MA, USA) and cross-linked with UV irradiation (UV Strata-linker1800, Stratagene, Cedar Creek, TX, USA). The membrane was probed with human albumin cDNA (ATCC# 59713, 1.0 kb) labeled by random priming using a commercial kit (Amersham Life Science) and [32^P] dCTP. The blot was prehybridized at 42℃ for 2 hours in 50% formamide, 0.1% SDS, 2 × Denhardt's solution, 5 × SSPE, and 0.1 mg/mL salmon sperm DNA. Probe (10⁶ cpm/mL) was added to the prehybridization solution, and the blot was hybridized for 16 hours at 42℃. The blot was then washed twice for 15 minutes at 55℃ in 2 × standard saline citrate and 0.1% SDS, and autoradiography was performed with Fuji X-ray film and an intensifying screen at -70℃. The albumin probe was then removed by boiling, and the same blot was rehybridized to a cDNA probe encoding the 36B4 ribosomal protein. Densitometric analysis was used to detect the intensity of the albumin and 36B4 signals.

Statistical comparisons between multiple groups were performed with an analysis of variance, and Bonferroni's method was applied to control for multiple testing. A p value < 0.05 was considered statistically significant.

Confluent Huh7 cells incubated for 24 hours secreted large amounts of albumin (5.18 ± 0.49 µg/mL). CsA significantly decreased the albumin release in a dose-dependent manner (p < 0.01). Vehicle (olive oil) did not affect the albumin concentration in conditioned medium. CsA inhibited albumin synthesis by 48%, 60% and 82% at concentrations of 10^-6 M, 10^-5 M, and 10^-4 M, respectively (Fig. 1A). The inhibition of albumin synthesis by CsA was statistically significant at 24 hours (Fig. 1B). A Western blot analysis for albumin in the conditioned medium released from Huh7 cells also revealed that CsA inhibited albumin synthesis in a dose-dependent manner at doses from 10^-7 M to 10^-5 M, whereas the vehicle did not (Fig. 1C).

Cell viability was tested by staining the cells from each experiment with trypan blue. More than 95% of cells treated with various concentrations of CsA were viable.

The Northern blot analysis revealed that CsA (10^-7-10^-5 M) did not inhibit albumin mRNA expression in Huh7 cells after a 24 hours incubation (Fig. 2A). In the time course, albumin mRNA expression in the untreated cells peaked between 2 and 4 hours and then decreased gradually until 24 hours. However, no difference in albumin mRNA expression was observed at any time point from 1 to 24 hours between untreated and CsA-treated Huh7 cells, indicating that inhibition of albumin synthesis by CsA occurred at the translational level (Fig. 2B).