Recently, orthotopic liver transplantation (OLTX) has become a standard therapy for patients with liver insufficiency, but this form of treatment is costly, complex, and limited by a scarcity of donor livers. A growing population of patients is likely to die waiting for a liver transplant because of a shortage of organs or because the patients are too sick to undergo OLTX. Consequently, there is a growing and compelling need to develop an attractive alternative to sustain patients with failing liver function. Currently, a bioartificial liver (BAL) is considered promising therapy for such patients. 1 Primary human hepatocytes seem to be a natural choice of cells for BAL; however, their relative scarcity is a major drawback, which is further compounded by the competing demands of OLTX. In addition, the lack of a regular supply of human livers makes planning for the appropriate clinical use of human hepatocytes difficult. Attempts to overcome this difficulty by maintaining long-term cultures of human hepatocytes have been hindered by both cost and the rapid loss of differentiated metabolic function. Using human hepatocytes that were isolated from liver tissue during partial hepatectomy or hepatocelluar carcinoma surgery has raised concerns about the transmission of infection or malignancy to the patient. The use of hepatocytes derived from animals raises the additional issues of ethical considerations, bioincompatibility between humans and animals, and transmission of pathogens. 2 Consequently, attention has been turned to using genetically modified human hepatocyte cell lines. We have made efforts to construct differentiated human hepatocyte cell lines using viral oncogenes. 3,4 Such cell lines could provide the advantages of unlimited availability, sterility, uniformity, and rapid preparation of human hepatocytes for BAL treatment. On the other hand, the loss of differentiated hepatic function has occurred to some extent in these oncogene transformed hepatocytes after increased passages. To overcome this problem, we have been exploring a strategy to enhance the differential phenotypes of such cells. It has been reported that expression of the sdi1 gene encoding a 21 kDa protein product, p21, is increased in senescent cells compared with their young counterparts. 5 The increase of cellular p21 gene expression has been well related to the onset of the senescent phenotype. p21sdi1 has been cloned as a gene encoding a cyclin dependent kinase (Cdk) inhibitor (Cip1) 6 and as a molecule transcriptionally activated by p53 (Waf1). 7 p21 has also been cloned as a melanocyte differentiation associated factor activated independent of p53 (mda-6). 8 These findings regarding p21 have elucidated the potent interactions of cell cycle regulation and cell differentiation. In the present study, we investigate the potential of exogenous p21 expression on differentiated hepatic phenotypes of simian virus 40 large T antigen (SV40Tag) immortalized human NKNT-3 cells.
Materials and Methods
Immortalization of Human Hepatocytes
Human hepatocytes were transduced with a retroviral vector SSR#69 encoding the SV40Tag gene, as was previously reported. 3 One of the resulting clones, NKNT-3, which grew steadily without any obvious crises and doubled in cell number in approximately 48 h, was used in the present study. NKNT-3 cells were maintained with the chemically defined serum-free CS-C medium (Cell Systems Co, Seattle, WA). The cell cultures were carefully examined by an inverted phase contrast microscope.
Status of Endogenous p21 Expression
To examine the endogenous expression of p21 in NKNT-3 cells, we performed immunofluorescent staining and Western blot analysis on the NKNT-3 cells. Total cell lysates, which had been obtained from an NKNT-3 culture, were prepared with a cell lysis buffer, resolved on 12% SDS-PAGE, and transferred onto nitrocellulose membranes (Amersham, Tokyo, Japan), as was previously reported. 9 Mouse monoclonal antibody to human p21 (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the samples, followed by a horseradish-peroxidase-conjugated anti-mouse IgG secondary antibody (1: 2000) (Medical and Biological Labs Corporation, Ltd.). Human β-actin protein served as an internal control. We then used an enhanced chemiluminescence system (ECL detection kit, Amersham) to detect human p21 and human β-actin. To determine the localization of endogenous p21 expression, immunofluorescence studies were performed on NKNT-3 cells, which were inoculated on coverslips and then placed on one well of a six well plate. A mouse monoclonal IgG antibody to human p21 (1:100) and a fluorescein isothiocyanate conjugated sheep polyclonal antibody to mouse IgG (1:2000) (Sigma-Aldrich, St. Louis, MO) were used for indirect immunofluorescent staining. The cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature (RT). Free aldehyde groups were quenched with 50 mmol/L NH4Cl in PBS for 10 min. The samples were washed three times with PBS at RT and made permeable with cold 100% ethanol for 30 min at 4°C. After three washes with PBS, the samples were incubated for 30 min at RT in blocking solution, which was made of 10% skim milk, 10% PBS, 0.1% sodium azide, and 0.1% octylphenol ethylene oxide condensate (Triton X-100, Sigma-Aldrich) in PBS. The cells were treated with a mouse monoclonal antip21 IgG in the blocking solution for 1 h at 37°C. After washing the samples three times with the blocking solution, the samples were treated for 1 h at 37°C with the secondary antibody. The cells were again washed three times with the blocking solution, and, for observation, the coverslips were mounted onto slides using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). A fluorescence microscope (Model Axiophot FL, Carl Zeiss, Inc, Oberkochen, Germany) was used to visualize the localization of p21.
Evaluation of Adenoviral Gene Transfer to NKNT-3 Cells
A replication-deficient recombinant adenovirus vector producing Escherichia coli LacZ reporter gene, Ad-Lacz (provided by Dr. Jack A. Roth, University of Texas, MD Anderson Cancer Center, Houston, TX), was used to evaluate the efficiency of adenoviral gene delivery to the NKNT-3 cells. The Ad-LacZ construct expressed the LacZ gene under the cytomegalovirus (CMV) promoter. NKNT-3 cells at 2 × 106 were plated on the six well plates and infected with Ad-LacZ at multiplicity of infections (MOIs) 1, 5, 10, and 25. For cell staining, 24 h after Ad-LacZ infection, the NKNT-3 cells were washed with PBS twice, fixed with 0.25% glutaraldehyde, and stained with 0.1% 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal). The X-gal positive cells were identified using a phase-contrast microscope.
Adenoviral Transfer of a p21 Gene to NKNT-3 Cells
A recombinant adenovirus vector encoding a human p21 cDNA derived by the CMV promoter, Ad-p21 (provided by Dr. Jack A. Roth), was constructed as previously characterized in detail. 10,11 As a control, Ad-LacZ was used in the present p21 study. The viral stocks were quantified by a plaque forming assay using 293 cells and then stored at −80°C before they were used.
Adenoviral Infection of NKNT-3 Cells with p21
NKNT-3 cells were transduced with Ad-p21 at MOI 10, and 48 h later, the cells were subjected to immunofluorescent staining and Western blot analysis for p21 expression using the procedure that was detailed above.
Effect of Adenoviral Infection of p21 on Cell Structures of NKNT-3 Cells
To examine the effect of Ad-p21 on cell structures, cellular distribution of global actin (G-actin) was evaluated in NKNT-3 cells that had been infected with Ad-p21 at MOI 10. Forty-eight hours after the Ad-p21 transduction, cells were subjected to immunofluorescent staining to detect G-actin expression, as described in the previously mentioned procedure. The cells were then treated with mouse monoclonal anti-G-actin IgG, followed by treatment with rabbit secondary antibody reacting to mouse IgG.
Western Blot Analysis for p-450 Associated Enzymes (CYPs) 3A4 and 2C9 of Ad-p21 Infected NKNT-3 Cells
Forty-eight hours after infection with Ad-p21 at MOI 10, total cell lysates were prepared from NKNT-3 cells, as described previously. 9 Samples were treated with a goat rabbit polyclonal antibody against human CYP 3A4 (1:100)(Daiichi Pure Chemicals Co, Ltd, Tokyo, Japan), a rabbit polyclonal antibody against human CYP 2C9 (1:100)(Daiichi Pure Chemicals Co, Ltd), and a horseradish-peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2000) (Medical and Biological Labs Co, Ltd). The protein expression of human β-actin was used to verify the equal loading of the samples. Human CYP 3A4 and CYP 2C9 were then detected using an ECL detection kit (Amersham). Human actin protein served as an internal control. The relative percentage of CYP 3A4 and CYP 2C9 to actin was calculated using the National Institutes of Health Image.
Flow Cytometric Analysis of NKNT-3 Cells after Ad-p21 Infection
To examine the cell cycle, NKNT-3 cells were subjected to flow cytometric analysis 48 h after transduction with Ad-p21 at MOI 10. Cells were trypsinized, washed twice with cold PBS, and resuspended in PBS containing 0.1% octylphenol ethylene oxide condensate and 0.1% ribonuclease for 5 min at RT. The samples were then stained with propidium iodide (0.1 mg/ml), filtered through a 40 μM pore size nylon mesh, and analyzed in a cell sorter (FACScan, Becton Dickinson, Mountain View, CA) for DNA content. Cell debris and fixation artifacts were gated out, and G0/G1, S, and G2-M populations were quantified using the ModFit LT program for Macintosh (Version 1.01, Verity Software House, Inc, Sunnyville, CA).
Endogenous p21 Expression in NKNT-3 Cells
Immunofluorescent staining and Western blot analysis were used to detect endogenous p21 expression in NKNT-3 cells. As shown by Hoechst nuclear staining and the immunofluorescence staining (Figure 1), endogenous p21 expression was almost absent in the NKNT-3 cells. Similarly, Western blot analysis showed no bands of p21 in the NKNT-3 cells.
Efficient Adenoviral Gene Transfer to NKNT-3 Cells
NKNT-3 cells were infected with an Ad-LacZ construct expressing LacZ gene under the CMV promoter. Twenty-four hours after Ad-Lacz infection, the NKNT-3 cells were stained with the X-gal detection solution. The LacZ positive cells were identified by phase-contrast microscopy. LacZ was detected in nearly 100% of the NKNT-3 cells after they had been infected with Ad-LacZ at MOI > 5. This shows that efficient adenoviral gene transfer can be performed in NKNT-3 cells.
Expression of p21 in Ad-p21 Infected NKNT-3 Cells
To detect p21 expression, an immunofluorescence study and a Western blot analysis were performed in NKNT-3 cells 48 h after Ad-p21 infection at MOI 10. The immunofluorescent staining showed nuclear localization of transduced p21 in the NKNT-3 cells (Figure 1), which was confirmed by Hoechst staining (Figure 1). No abnormality in G-actin distribution in the Ad-p21 infected NKNT-3 cells was detected, indicating that the adenoviral transduction of p21 did not affect cell structures (Figure 1). The Western blot analysis showed intense bands of p21 in Ad-p21 infected NKNT-3 cells. These results were not obtained in a control Ad-LacZ infection.
Morphologic Alterations of NKNT-3 Cells After Ad-p21 Infection
After the NKNT-3 cells had been infected with Ad-p21 at MOI 10, the morphologic changes of the cells were carefully examined using phase contrast microscopy. Following Ad-p21 transduction, decreased cell density and enlargement of cell size with lower nuclear to cytoplasmic ratios were observed in the NKNT-3 cells (Figure 2). These findings were not detected in Ad-LacZ infected NKNT-3 cells.
Expression of CYP 3A4 and CYP 2C9
To evaluate the protein expression of CYP 3A4 and CYP 2C9, Western blot analysis was used on 30 μg of protein that had been taken from Ad-p21 transduced NKNT-3 cells. As shown in Figure 3, more intense bands for CYP 3A4 and 2C9 were detected in Ad-p21 infected NKNT-3 cells than were detected in uninfected cells. Compared with unmodified cells, 1.6 and 1.4 times more CYP3A4 and CYP2C9, respectively, were detected in Ad-p21 infected NKNT-3 cells (Figure 3). These findings were not found in NKNT-3 cells that had been infected with Ad-Lacz.
Cell Cycle Analysis
After Ad-p21 infection, the development of the NKNT-3 cells was arrested in the G0/G1 phase. This did not occur in the Ad-LacZ infected NKNT-3 cells. These findings were consistent with the differentiated hepatic phenotype that had been induced in NKNT-3 cells following Ad-p21 transduction.
The shortage of donor livers for hepatocyte isolation limits the clinical application of hepatocyte based hybrid bioartificial livers. An attractive solution would be to use a cell line that could provide consistent and uniform cell preparation with sufficient quantity and adequate quality. Orthotopic liver transplantation (OLTX) has been the only therapeutic modality that significantly improves the prognosis of patients with failing liver function. However, liver replacement for a potentially reversible lesion is an aggressive form of therapy and requires life-long immunosuppression. A growing population of patients is likely to die waiting for OLTX. There is an expanding need for an attractive alternative to OLTX, for a way to sustain patients with liver insufficiency while waiting for a donor liver or in case the transplanted liver fails, and a way to improve the survival of patients in whom OLTX is not an option. With the current remarkable advances in biotechnology, a bioartificial liver support system represents an attractive option for such patients. 1 On the other hand, considering the cost of hepatocyte isolation and the need for immediate availability of consistent and functionally uniform cell preparations, it is unlikely that human hepatocytes could be isolated on a large enough scale to treat more than a fraction of the patients who need BAL therapy. The use of a human hepatocyte cell line would be a promising alternative to primarily isolated hepatocytes. In an attempt to test the feasibility of this approach, we have immortalized human hepatocytes with SV40Tag and established an NKNT-3 cell line. Such technology would allow for unlimited availability of human cells with an adequate quantity for cell based biologic therapies; however, long-term in vitro cultivation of immortalized cells with continued existence of viral oncogenes often results in the loss of differentiated phenotypes after increased passages. Enhancement or reversion of differential phenotypes is of extreme importance in such cells for the development of hybrid bioartificial organs.
We have focused on the molecule p21, a potent cyclin-dependent kinase inhibitor. 5–8 Also known as sdi1, Cip1, WAF1, or mda-6, p21 was originally identified as the molecule that regulates the transition from the G1 phase to the S phase of the cell cycle. During skeletal muscle differentiation, the muscle-specific transcriptional regulator myogenic differentiation increases p21 expression, thereby inducing terminal cell cycle arrest. 12 Moreover, it has been reported that p21 protein expression is more frequently detected in well differentiated non-small cell lung carcinoma than in poorly differential tumors. 13 Overexpression of p21 has been shown to induce differentiation in monoblastic cell lines. 14 Together, these observations suggest that p21 is involved in a terminal differentiation program in both normal and cancer cells. The effect of p21 on immortalized human hepatocytes, however, has not been well documented.
Adenovirus mediated gene transfer is a highly efficient gene delivery system in various cells. In the present study, we used a recombinant adenovirus to overexpress p21 in immortalized human hepatocyte NKNT-3 cells. Preliminary titration studies showed that transient transgene expression in most populations of infected cells of various types could be expected at up to MOI 100. 11 In fact, strong induction of exogenous p21 expression was achieved in NKNT-3 cells, and the transduced p21 protein was localized in the nucleus. The p21 protein has an intrinsic nuclear localizing signal that could facilitate nuclear entry of associated cyclin E-Cdk2 complexes, as well as cyclin D-Cdk4 complexes. 15
Transduced p21 blocked the cell cycle progression and caused differentiation-specific morphologic changes in NKNT-3 cells. Although the active regions of p21 that bind to Cdk as well as proliferating cell nuclear antigen 16,17 have been identified, p21 itself is not likely to be a transcriptional factor. Therefore, transcriptional activation of the CYP 3A4 and CYP 2C9 promoters by p21 gene transfer may be indirect, presumably through other transcriptional factors, and the precise mechanism of this stimulatory effect remains to be identified. Our flow cytometric analysis of cell cycle distribution revealed that Ad-p21 infected NKNT-3 cells were arrested in the G0/G1 phase.
In summary, our results demonstrate that introduction of the p21 gene into human immortalized hepatocytes can increase CYP 3A4 and CYP 2C9 expression and induce the differentiated phenotype. Therefore, exogenous expression of p21 in such cells may be an attractive strategy for cell-based biologic therapies.
This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan, and by grants from the Ministry of Economy and Industry, Japan. We would like to thank Dr. Jack A. Roth (Department of Thoracic and Cardiovascular Surgery, University of Texas, MD Anderson Cancer Center, Houston, TX) for the use of adenovirus vectors Ad-p21 and Ad-LacZ.
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