We established two independent lines of primary and immortalized porcine fibroblast cells from miniature (MPF cells) and domestic (PF cells) pigs, respectively (Figure 1A). In the presence of 10% FBS, the growth rate of the early passage PF cells (P3) was shown to be more rapid than that of the MPF cells (P3), whereas both cells failed to proliferate in the presence of 0.5% FBS, thereby indicating that both cells require FBS for proliferation (Figure 1B). In contrast to primary cells, immortalized MPF cells (P60) grew faster than immortalized PF cells (P60) (Figure 1C), implying possible genetic alterations that enable to change in cellular growth property during immortalization process. We next determined the in vitro lifespan of primary MPF and PF cells using a standard 3T3 protocol. As shown in Figure 2A, there was a marked difference in the growth curves of the MPF and PF cells. For MPF cells, replicative senescence appeared at two distinct passages (P5 and P28) as judged by a flat morphology and senescence-associated β-galactosidase activity (SA-β-gal) (Figure 2B and 2C), and a growth rate was shown to decrease until passage 34, but rapidly increase after passage 35. It is noteworthy that less than 35% of MPF cells at passage 7 through passage 24 displayed SA-β-gal-positive (Figure 2B and 2C, data not shown). For the PF cells, the replicative senescence appeared at two distinct passages (P13 and P38). Taken together, the MPF cells grew at a slower rate and had a shorter in vitro lifespan compared to the PF cells. Since the MPF and PF cells, after passages 35 and 40, respectively, grew continuously without a detectable senescent phenotype, we speculate that both the MPF and PF cells by passage 60 would be spontaneously immortalized.

Since it has been well documented that p53 gene, a cell cycle checkpoint and tumor suppressor, was commonly inactivated in numerous immortal and transformed cells 22, we determined expression levels of p53 and p21^WAF1, one of p53-downstream target genes, as well as biological function of p53 in the primary and immortalized MPF and PF cells. As shown in Figure 3A, expression level of p53 protein was found to be similar in the primary and immortalized PF cells, while p21^WAF1 was relatively upregulated in the immortalized PF cells compared to the primary PF cells. However, we think that expression levels of p53 and p21^WAF1 proteins might be more elevated in the immortalized PF cells compared to primary PF cells as normalized to α-tubulin level (P3 and P60 in Figure 3A). When primary PF cells were treated with doxorubicin (a DNA-damaging agent) that enables to stabilize and activate p53 protein, p53 protein level was shown to be markedly elevated with concomitant increase of p21^WAF1 (Figure 3B). Interestingly, the expression of p21WAF was relatively decreased in the immortalized PF cells treated with doxorubicin (Figure 3B).

However, like other immortalized or transformed cells, the expression of p53 protein was shown to be markedly elevated in the immortalized MPF cells, whereas p21^WAF1 protein was dramatically downregulated in these cells as compared to the primary MPF cells (Figure 3A). In addition, when primary MPF cells were treated with doxorubicin, p53 protein was shown to be markedly elevated in these cells with concomitantly slight increase of p21^WAF1, whereas expression of the p53 protein was not changed by a DNA damage response, and p21^WAF1 protein was barely detectable in the immortal MPF cells, regardless of doxorubicin (Figure 3B). Furthermore, in the presence of doxorubicin (3 and 5 uM), the immortalized MPF cells were more resistant to cell death as compared to primary MPF cells (p < 0.05, Figure 3C), whereas immortalized PF cells showed slightly increased resistance to doxorubicin (only at 5 uM) as compared to primary PF cells (Figure 3C). Taken together, these results indicate that immortalized MPF cells, but not immortalized PF cells, may be defective for the p53 regulatory function.

To address whether primary and immortal MPF and PF cells have a transformed property, we studied the anchorage-independent growth of primary MPF and PF cells by growing the cells in a soft-agar culture condition. As shown in Figure 4A, immortal MPF (passage 60), but not immortal PF cells, were enabled to grow in the soft-agar, suggesting that the immortalized MPF cells should possess a transformed property. As determined chromosome abnormality of the primary and immortal MPF and PF cells by a karyotyping (Figure 4B), 81 of the 100 immortalized MPF cells have abnormal chromosome numbers of 2N = 57.2 on average, while all of primary MPF and PF as well as immortalized PF cells were shown to have normal chromosome number of 2N = 38 on average (data not shown).

Cellular immortalization and transformation have been shown to associate with loss of p53 and gain of telomerase activity 2324. Therefore, we transduced SV40LT (one of the p53 inactivators, 25) and hTERT (human telomerase catalytic subunit) into primary MPF and PF cells in order to determine a possible causal role of the activated p53 or telomerase activity for a shorter lifespan and earlier senescent-mediated growth arrest of the primary MPF cells as compared to primary PF cells. Growth rate of the SV40LT- or hTERT-transduced PF cells was shown to be faster than that of the SV40LT- or hTERT-transduced MPF cells in the presence of 10% FBS, whereas these cells failed to proliferate in the presence of 0.5% FBS (Figure 5A and 5B). In contrast, the SV40LT+hTERT-transduced MPF cells were shown to proliferate relatively faster than SV40LT+hTERT-transduced PF cells in the presence of 10% FBS, whereas these cells failed to grow in the presence of 0.5% FBS (Figure 5C). In examining their in vitro lifespan by the 3T3 protocol, both SV40LT-transduced MPF and PF cells proliferated continuously and without any detectable senescent phenotype (Figure 6A), whereas transduction of hTERT into primary MPF and PF cells failed to extend their in vitro lifespan (Figure 6B). Interestingly, the hTERT-transduced MPF and PF cells were shown to become senescent at passages 9 and 13, and to enter crisis stage from passages 10 and 14, respectively. Meanwhile, transduction of SV40LT+hTERT into primary MPF and PF cells, like transduction of SV40LT alone, exhibited an extension of their in vitro lifespan (Figure 6C). Of interest, the growth rate of the SV40LT+hTERT-transduced MPF cells were shown to be faster than their counterpart PF cells. PF and MPF cells transduced with SV40LT or SV40LT+hTERT have continuously proliferated beyond passage 60 without any detectable senescent barriers and we are of the opinion that these cells should be immortalized.

Since hTERT, distinct from SV40LT, failed to extend cellular lifespan, we examined expression and relative activity of hTERT in the control and hTERT-transduced cells. Although hTERT mRNA was found to be expressed as determined by RT-PCR, the hTERT activity did not increase in the hTERT-transduced cells as compared to nontransduced cells (data not shown). Furthermore, we found that endogenous telomerase activity in the primary and immortal MPF and PF cells was similar to that in the primary human BJ fibroblast cells (data not shown). These results suggest that activation of the telomerase might not be necessary for cellular immortalization at least in the MPF cells of this study, whereas loss of p53 should be sufficient for immortalization and p53 activation in these cells might be a major determinant for the replicative senescence.

Göttingen miniature pigs were used as the miniature pig strain, while the three-way crossbred pig (Duroc, Landrace and Yorkshire) that is commercially used for pork production was our domestic strain. The Göttingen strain miniature breed was developed at Göttingen University in the 1960s by the cross-breeding of the Minnesota miniature pig initially with the Vietnamese pot-bellied pig and followed by the German Landrace pig which yielded pale skin animals. The Göttingen strain is a white non-pigmented small-sized miniature pig with an adult body weight of 30–40 kg 54. All animals received humane care in compliance with the guide for the care and use of laboratory animals 55.

Porcine fibroblast cells isolated from the ears of two 1-day-old female miniature pigs (MPF) and two 1-day-old female domestic pigs (PF) were grown and maintained in DMEM/high glucose (Hyclone) media enriched with 10% FBS (Hyclone), 1% penicillin-streptomycin (Gibco), and 2 mM L-glutamine (Gibco). To determine cellular lifespans in this study, primary MPF and PF cells were plated at a density of 3 × 10⁵ cells/10 cm dish and passaged every 3 days following the standard 3T3 protocol; the number of population doublings per day was calculated.

Cell growth and growth rates were determined by plating cells at a density of 1 × 10⁴ cells in 10% FBS and 2.5 × 10⁴ cells in 0.5% FBS in 6-well plates and staining with a 0.01% crystal violet solution every other day for 6 days. Crystal violet was extracted from the stained cells using 10% acetic acid and subjected to spectrophotometric analysis (595 nm) to determine relative cell growth rates 56. To assess responses to DNA damage, relatively early passage (P4) and late passage (P60) cells were treated with doxorubicin (0, 1, 3, and 5 uM)) for 24 h, and their viability measured by the standard trypan blue exclusion method.

To conduct the senescence-associated β-galactosidase activity assay, cells were fixed with 0.5% glutaraldehyde (pH 7.2) and washed in PBS (pH 7.2) supplemented with 1 mM MgCl₂ and then stained in X-gal solution (1 mg/mL X-gal, 0.12 mM K₃Fe [CN]₆, 0.12 mM K₄Fe [CN]₆, 1 mM MgCl₂ in PBS at pH 6.0) overnight at 37°C. After washing with phosphate-buffered saline, the plates were viewed by light microscopy.

Cells were infected with replication-defective retroviruses produced by a PT67 amphotropic packaging cell line (Clontech) that had been transfected with pBabe-SV40LT-puro vector. Supernatants of the stable transfected PT67 cells (>70% confluent) were filtered through a 0.45 um filter to remove cellular debris and used to infect the cells three times at 12 h internals. The infected cells were selected with 2 ug/mL puromycin (Clontech) for 7 days. Cells were transfected with the pCI-hTert-neo vector and selected with 500 ug/mL neomycin (G418, Gibco) for 2 weeks.

Cell extracts were prepared using a RIPA lysis buffer containing 1× protease inhibitor cocktail (Roche). Proteins in the cell extracts (50 ug) were separated on a 4~12% pre-cast SDS-PAGE gradient (Invitrogen) and transferred to PVDF membranes (Millipore). The membranes, which had been blocked with 5% skim milk, were incubated with anti-p53 antibody (sc-126, Santa Cruz Biotechnology, 1:500 dilution), anti-p21^WAF1 antibody (sc-397, Santa Cruz Biotechnology, 1:200 dilution), and anti-α-tubulin antibody (T9026, sigma, 1:5000 dilution), followed by incubation of HRP conjugated anti-mouse IgG and anti-rabbit IgG secondary antibody (Pierce). The immunoblot signals were detected with a SuperSignal West Pico kit (Pierce).

To measure cell anchorage independence, the primary MPF and PF cells (1 × 10⁴) were cultured in 6-well soft-agar dishes (1.6% and 0.7% bottom and top agar, respectively) for 3 weeks.

The mitotic chromosomes of MPF and PF cells were obtained following standard methods with slight modifications. Thus, cells were initially plated in 10% FBS containing DMEM for 48~72 h, treated with 0.01 mg/mL colcemid (Gibco), lysed for 15 min in a hypotonic solution, and fixed in a methanol:acetic acid (3:1) solution. Chromosomes were stained with 4% Giemsa solution in Gurr's buffer and the number of chromosomes in metaphase (n = 100 cells) from each cell line were determined.

All experiments were replicated three times at least. Statistical significance was assessed by ANOVA, followed by Duncan's test in SAS software package (Ver.9.1).