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. 2015 Jan 27;112(4):1053-8.
doi: 10.1073/pnas.1418203112. Epub 2014 Dec 30.

INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform

Xiao Tian  1 , Jorge Azpurua  1 , Zhonghe Ke  1 , Adeline Augereau  2 , Zhengdong D Zhang  3 , Jan Vijg  3 , Vadim N Gladyshev  2 , Vera Gorbunova  4 , Andrei Seluanov  4
Affiliations

INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform

Xiao Tian et al. Proc Natl Acad Sci U S A. .

Abstract

The naked mole rat (Heterocephalus glaber) is a long-lived and tumor-resistant rodent. Tumor resistance in the naked mole rat is mediated by the extracellular matrix component hyaluronan of very high molecular weight (HMW-HA). HMW-HA triggers hypersensitivity of naked mole rat cells to contact inhibition, which is associated with induction of the INK4 (inhibitors of cyclin dependent kinase 4) locus leading to cell-cycle arrest. The INK4a/b locus is among the most frequently mutated in human cancer. This locus encodes three distinct tumor suppressors: p15(INK4b), p16(INK4a), and ARF (alternate reading frame). Although p15(INK4b) has its own ORF, p16(INK4a) and ARF share common second and third exons with alternative reading frames. Here, we show that, in the naked mole rat, the INK4a/b locus encodes an additional product that consists of p15(INK4b) exon 1 joined to p16(INK4a) exons 2 and 3. We have named this isoform pALT(INK4a/b) (for alternative splicing). We show that pALT(INK4a/b) is present in both cultured cells and naked mole rat tissues but is absent in human and mouse cells. Additionally, we demonstrate that pALT(INK4a/b) expression is induced during early contact inhibition and upon a variety of stresses such as UV, gamma irradiation-induced senescence, loss of substrate attachment, and expression of oncogenes. When overexpressed in naked mole rat or human cells, pALT(INK4a/b) has stronger ability to induce cell-cycle arrest than either p15(INK4b) or p16(INK4a). We hypothesize that the presence of the fourth product, pALT(INK4a/b) of the INK4a/b locus in the naked mole rat, contributes to the increased resistance to tumorigenesis of this species.

Keywords: INK4; naked mole rat; p15; p16.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The INK4a/b locus of the NMR produces an alternative protein isoform pALTINK4a/b composed of the first exon of p15INK4b and the second and third exons of p16INK4a. (A) Schematic of the INK4a/b locus showing the location of the primers used to amplify the three INK4a/b transcripts. (B) RT-PCR products corresponding to the three INK4a/b transcripts amplified from RNA isolated from three lines of naked mole rat fibroblasts, with primers shown in A and Table S2; pALTINK4a/b was amplified with primers p15-5UTR and p16-E2. NSF2 mut is the line of spontaneously mutated naked mole rat fibroblasts that lost INK4a/b locus expression and do not show ECI; NSF8 are primary naked mole rat skin fibroblasts; NEF9 are naked mole rat embryonic fibroblasts. (C) Sequences of the Exon1/Exon2 splice junctions in p15, p16, and pALT. (D) RT-PCR showing INK4a/b products amplified from naked mole rat tissues. GAPDH was used to show that equal amounts of RNA template were used for each tissue. The reactions were repeated at least three times, and representative gels are shown.
Fig. 2.
Fig. 2.
pALTINK4a/b expression is induced by ECI, confluence, and senescence. (A) Naked mole rat fibroblasts were passaged under four different conditions. “Growing” cells were passaged at low cell density to ensure continuous cell proliferation. “HAase” cells were passaged at low cell density in the presence of hyaluronidase. “ECI” cells were kept in the same plate without passaging for 10–14 d until they stopped proliferation and entered the ECI state. “Confl.” cells were cultured in the presence of hyaluronidase until they reached confluence at high cell density. RNA was extracted from the cells, and expression from INK4a/b locus was analyzed by qPCR. (B) Naked mole rat cells were treated with indicated doses of UV-C and analyzed for expression of INK4a/b products as described above. (C) Naked mole rat cells were treated with 20 Gy of γ-irradiation, kept for 30 d until they developed senescent morphology, and analyzed for expression of INK4a/b products as described above. Four lines of low-passage naked mole rat skin fibroblasts were used for each experiment. Error bars indicate SD; *P < 0.05; **P < 0.005; Student’s t test.
Fig. 3.
Fig. 3.
pALTINK4a/b expression is induced by oncogenic stimuli. (A) Naked mole rat cells were subjected to anchorage-independent growth to trigger anoikis by placing them in plates precoated with 1% agarose for 48 h. RNA was extracted from the cells, and expression from INK4a/b locus was analyzed by qPCR. (B) Naked mole rat cells were transfected with plasmids encoding HRas V12 or BRAF E600 oncogenes or pControl plasmid encoding GFP. RNA was extracted 10 d after transfection, and expression from INK4a/b locus was analyzed by qPCR. Four lines of low-passage naked mole rat skin fibroblasts were used for each experiment. Error bars indicate SD; *P < 0.05; **P < 0.005; Student’s t test.
Fig. 4.
Fig. 4.
pALTINK4a/b overexpression triggers cell-cycle arrest. Naked mole rat cells were transfected with constructs expressing HPRT (control), p15INK4b, p16INK4a, or pALTINK4a/b under the CMV promoter. (A) Expression of the INK4a/b products was verified with RT-PCR. (B) Microphotograph showing cells transfected with p15INK4b, p16INK4a, or pALTINK4a/b 6 d after transfection. Control cells continued to proliferate whereas cells transfected with INK4a/b products entered cell-cycle arrest. (C) Cell-cycle arrest induced by expression of p15INK4b, p16INK4a, or pALTINK4a/b was quantified at indicated times after transfection by staining cells with propidium iodide and quantifying the number of cells in S-phase by FACS analysis. The experiments were repeated three times, and error bars show SD; statistical significance is indicated by asterisk. Three lines of low-passage naked mole rat skin fibroblasts were used for each experiment. *P < 0.05; **P < 0.005; ***P < 0.0005, Student’s t test.
Fig. 5.
Fig. 5.
pALTINK4a/b overexpression attenuates γ-irradiation–induced apoptosis in NSF2 mut cells. NSF2 mut cells were transfected with plasmids encoding HPRT (control), p15INK4b, p16INK4a, or pALTINK4a/b under the CMV promoter. Two days after transfection, cells were subjected to 20 Gy γ-irradiation. (A) At four and 10 d after γ-irradiation, the cells were harvested, and the percent of apoptotic cells was analyzed by Annexin-V staining and flow cytometry. (B) Cells were photographed 10 d after γ-irradiation. Control plate shows many floating apoptotic cells whereas plates transfected with INK4a/b products show lower numbers of apoptotic bodies and many growth-arrested cells. The experiments were repeated three times, and error bars show SD; statistical significance is indicated by asterisk. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005, Student’s t test.
Fig. 6.
Fig. 6.
Structure of the naked mole rat INK4a/b locus. The diagram shows the genomic locus and the canonical p15INK4b, p16INK4a, and pARF transcripts, as well as the pALTINK4a/b transcript unique to the naked mole rat.
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