TP53INP1-deficient mice were generated as described previously (18). At 14.5 days post coitum, TP53INP1^−/−-MEF and TP53INP1^+/+-MEF were prepared from embryos derived from the homozygous breeding of TP53INP1-deficient mice and of their wild-type littermates, according to earlier reports in the literature (12,14,19). These cells were transformed and immortalized by transduction with the pBabe-E1A/rasV12 retroviral vector, encoding the constitutively active ras. The TP53INP1 genotypes of the respective MEFs were determined by polymerase chain reaction (PCR). Genotype analyses were conducted on genomic DNA from MEF, with the following PCR primers F, R1 and R2: F, 5′-AATGTATGCAATCTTAGCTGA TGC-3′; R1: 5′-TCTTGAGGTAACATAGTGAAATGC-3′; and R2: 5′-CCAAACACTGTCACTGTATTGATA-3′. PCR analysis was performed as described in an earlier study (18).

TP53INP1^−/−-MEF and TP53INP1^+/+-MEF were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) Glutamax medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen Corp., Groningen, The Netherlands) at 37°C under 5% in CO₂ atmosphere. The ras-transformed MEFs at early passages were used for subsequent experiments.

TP53INP1^−/−-MEF and TP53INP1^+/+ MEF were treated with 0, 5, 10 and 20 ng/ml gemcitabine (Eli Lilly and Co., Indianapolis, IN, USA) for 24 h.

Whole cell lysates were prepared using RIPA buffer with a protease inhibitor cocktail (Roche Diagnostics KK, Basel, Switzerland). A 25 μg aliquot of each cellular protein sample was diluted in loading buffer (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). The proteins were separated in sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P polyvinylidene fluoride 7 (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Subsequent to blocking with 5% skimmed milk solution, the membrane was treated with monoclonal antibodies against p21, p53, TP53INP1 (Abcam, Cambridge, UK), Rb (Cell Signaling Technology, Inc., Beverly, MA, USA), and β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA). A Cell Cycle/Checkpoint Antibody Sampler kit (Cell Signaling Technology, Inc.) was also used. The respective bound antibodies were detected with an IgG rabbit monoclonal antibody (SouthernBiotech, Birmingham, AL, USA), then visualized with Immune Star LD (Wako Pure Chemical Industries, Ltd., Osaka, Japan), using Fuji Xerox LAS4000 image analyzer (Fujifilm Corp., Tokyo, Japan). The intensity of each protein signal was measured using Multi Gauge^® (Fujifilm Corp.).

Total RNA was extracted from TP53INP1^−/−-MEF and TP53INP1^+/+-MEF, using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The RT reaction was conducted with a SuperScript™ III First-Strand Synthesis System (Invitrogen Corp.) using random hexamers to generate complementary DNA (cDNA) according to the manufacturer’s instructions. RT-PCR was conducted using TaqMan probe; TaqMan^® Fast Universal PCR Master mix (Applied Biosystems, Foster City, CA, USA). Sequences of the primers were CDKN1A (p21) mixed primers; Mm00432448_m1 (Applied Biosystems), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mixed primers; Mm99999915_g1 (Applied Biosystems). RT-PCR was conducted, using a Real-Time PCR system (Applied Biosystems 7900HT Fast; Applied Biosystems) as well as the ΔΔCT method. The result was analyzed with the SDS software ver. 2.3 (Applied Biosystems).

The same experiments were performed 3 times. The mean number of cells in each experiment was calculated with standard deviations (SDs).

The cells at the exponential growth phase were labeled with 5-bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich Corp.) [1 mM dissolved in phosphate-buffered saline (PBS)] in a final concentration of 10 μM in the medium. BrdU labeling and subsequent handling of the cells were conducted under subdued light. Subsequent to 15 min of incubation at 37°C, the BrdU-containing medium was removed, and the cells were rinsed twice with PBS. The labeled cells were trypsinized and counted, using a hemocytometer and centrifuged at 5,000 × g for 5 min at 4°C. The pelleted cells were suspended and fixed in ice-cold 70% ethanol (∼2×10⁶ cells/ml) and stored at −20°C until analysis.

The fixed cells were resuspended in 1 ml PBS containing 0.5% Triton X-100, treated with 2N-HCl for 30 min at room temperature and neutralized with 0.1 M sodium tetraborate (pH 8.5). Subsequent to washing twice with washing solution [PBS containing 0.5% Tween-20 and 1% bovine serum albumin (BSA)], the cells were stained with fluorescein isothiocyanate (FITC)-labeled mouse anti-BrdU antibody or FITC-labeled mouse IgG1κ isotype as a negative control (both antibodies included in FITC Mouse Anti-Human BrdU set; Becton-Dickinson, Franklin Lakes, NJ, USA) overnight at 4°C. The stained cells were washed twice with 1 ml ice-cold PBS and incubated with 0.25% RNase (Sigma-Aldrich Corp.) for 30 min at room temperature. The cells were then stained with 5 μg/ml propidium iodide (PI; Sigma-Aldrich Corp.) and examined using FCM (FACSCalibur; Becton-Dickinson) for green and red fluorescence, which determined the relative number of BrdU-labeled cells and cells examined, respectively.

A total of 200 exponentially growing TP53INP1^−/−-MEFs or INP1^+/+-MEFs were seeded on a 6-cm dish and grown in DMEM Glutamax medium, containing 10% FBS at 37°C in a 5% CO₂ atmosphere. After a week, the colonies per dish were counted; each cell line was counted immediately.

When cells were confluent, gemcitabine was added to the medium 3 days subsequent to cell seeding. TP53INP1^−/− and TP53INP1^+/+ cells were treated with 0, 5, 10 and 20 ng/ml gemcitabine for 24 h. Cell growth was determined using the colony formation assay. The same experiments were performed 3 times. The mean number of colonies in each experiment was calculated.

Data were analyzed using the Student’s t-test. Values were given as the mean ± SEM and P<0.05 was considered to indicate a statistically significant difference. The results were analyzed using Excel (Microsoft Corp.).

The TP53INP1, a nuclear localization protein gene, was amplified using PCR. Null mutation mice were generated by genotyping (18). As confirmed by PCR (Fig. 1A) and western blot analysis, TP53INP1 was deficient in TP53INP1^−/- MEFs, but expressed in TP53INP1^+/+-MEFs, respectively (Fig. 1B).

TP53INP1^−/−-MEFs and TP53INP1^+/+-MEFs were cultured and the colonies were confirmed. Subsequently, several subclones were established from the TP53INP1^−/−-MEFs and TP53INP1^+/+-MEFs. Two subclones from cells of each type (subclones a and b from the TP53INP1^−/−-MEFs; subclones c and d from the TP53INP1^+/+-MEFs) were selected.

Cell proliferation was measured by colony formation assay. The proliferation of TP53INP1^−/−-MEFs tended to be faster compared to TP53INP1^+/+-MEFs (a, b and c, d, respectively) (Fig. 2).

Cells were treated with gemcitabine for 24 h, then cell growth was assayed. Gemcitabine inhibited a significantly greater TP53INP1^−/− cell growth compared to TP53INP1^+/+ cells, in a concentration-dependent manner (Fig. 3) (P<0.05). According to the curve indicating the survival rate, IC₅₀ of TP53INP1^−/− was estimated as 2–3 ng/ml, while that of TP53INP1^+/+ was 5–10 ng/ml.

TP53INP1^−/−-MEFs and TP53INP1^+/+-MEFs were treated with gemcitabine at 10 ng/ml for 24 and 48 h. Subsequent to treatment, each cell cycle status was determined using FCM. Differences in each peak of G1, S and G2/M of TP53INP1^−/−-MEF and TP53INP1^+/+-MEF were impossible to confirm based on the results of FCM. Gemcitabine treatment generated almost equal apoptosis in both cell lines.

A previous report (16) demonstrated that TP53INP1 overexpression induced p21 expression, while findings of the present study confirmed the decrease of the mRNA and protein expressions induced by the TP53INP1 knockout. Moreover, another study described that oxidative stress decreased the p21 expression in TP53INP1 knockout cells; thus, the change in p21 expression was examined using gemcitabine treatment.

Subsequent to gemcitabine treatment in both cell lines, in RT-PCR, the p21 mRNA expression was significantly decreased in the TP53INP1^−/−-MEFs compared to TP53INP1^+/+-MEFs (P<0.05) (Fig. 4).

Subsequent to the same treatment in both cell lines, western blot analysis revealed that the protein expression of p21 was significantly lower in TP53INP1^−/−-MEFs compared to TP53INP1^+/+-MEFs (P<0.05) (Fig. 5).

It is already widely acknowledged that p53 is located upstream of p21. A previous study described that a decreased p21 expression resulted from a decreased p53 expression (21). In TP53INP1^−/− cells, the p21 expression decreased in RNA and protein. Whether or not these results were derived from the status of p53 expression was investigated. The expression of p53 was verified by western blotting in both TP53INP1^−/−-MEF and TP53INP1^+/+-MEF. This experiment demonstrated that p53 expression was not decreased in TP53INP1^−/−-MEFs (Fig. 6).