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[Cancer Research 65, 3249-3256, April 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Mouse p10, an Alternative Spliced Form of p15INK4b, Inhibits Cell Cycle Progression and Malignant Transformation

Ignacio Pérez de Castro1,2, Marta Benet1, María Jiménez3, Saba Alzabin1, Marcos Malumbres2 and Angel Pellicer1

1 Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, New York; 2 Molecular Oncology and 3 Experimental Therapeutics Programs, Centro Nacional de Investigaciones Oncologicas CNIO, Madrid, Spain

Requests for reprints: Ignacio Pérez de Castro, Molecular Oncology Program, Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, Madrid 28029, Spain. Phone: 34-91-224-6900; Fax: 34-91-732-8033; E-mail: iperez{at}cnio.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The INK4 family of proteins negatively regulates cell cycle progression at the G1-S transition by inhibiting cyclin-dependent kinases. Two of these cell cycle inhibitors, p16INK4A and p15INK4B, have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16INK4A, the INK4A locus encodes a splice variant, termed p12—specifically expressed in human pancreas—and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15INK4B tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15INK4B, p10 expression is also induced by oncogenic insults and transforming growth factor-ß treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulation of cell division is a highly complex process that involves many different pathways. During the last decade, many molecules implicated in cell cycle regulation have been identified (1). Cell cycle regulators include cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors, which are involved in the regulation of cell cycle progression in response to mitogenic and antimitogenic signaling. Progression throughout the cell cycle depends on the sequential activation of CDKs by their cyclin partners. Thus, complexes formed by CDK4 or CDK6 and D-type cyclins phosphorylate the retinoblastoma protein, thereby helping to cancel its growth-repressive functions at the G1-S cell cycle checkpoint. CDK4 and CDK6 activities are specifically inhibited by the INK4 family of CDK inhibitors. INK4 family members include p16INK4A, p15INK4B, p18INK4C, and p19INK4D, and are characterized by the presence of multiple ankyrin repeats, which participate in CDK binding.

The importance of the cyclin D/CDK4/INK4/retinoblastoma pathway in cancer is undeniable, since tumor-associated alterations in at least one of the proteins of this pathway have been found in >80% of all human malignancies (2). Alterations include translocation, amplification, or overexpression of D-type cyclins or CDKs, mutations in CDK4 and CDK6 (resulting in loss of INK4 binding), as well as inactivation of retinoblastoma or the CDK inhibitors (mainly p16INK4A and p15INK4B). The importance of the INK4A locus in cance r was further underscored after the discovery of an additional protein, p19ARF (p14ARF in humans), as a tumor suppressor encoded by an alternative reading frame (3). Whereas cell cycle inhibition by p16INK4A is mediated by retinoblastoma activation, it was soon realized that ARF exerts its tumor suppression functions through the p53 pathway since ARF stabilizes p53 by negatively regulating mdm2 (4–6). In fact, lack of expression of ARF has been associated with several tumor types (7, 8) and its deletion produces a tumor-prone phenotype in gene-targeted mice (9). Another splice variant of INK4A, termed p12, is specifically expressed in human pancreas (10). This protein does not interact with CDK4 but is capable of suppressing growth in a retinoblastoma protein–independent manner.

Similarly, an alternatively spliced form, termed as p10, has been described in the human INK4B locus (11). Like p12, human p10 is synthesized through the use of an alternative splice donor site within intron 1 and retains growth-inhibitory activity despite undetectable CDK4 and CDK6 binding. However, unlike p12, which is specifically expressed in pancreas, human p10 is present in several different normal and tumor human cell lines (11).

Here, we describe a functional characterization of the mouse orthologue of human p10. Like its human counterpart, mouse p10 arises from the use of an alternative splice donor site within intron 1 of the INK4B locus. Mouse p10 is widely expressed in murine tissues although to a lesser extent than p15. Like p15INK4B, p10 expression is also induced by oncogenic insults and transforming growth factor-ß (TGF-ß) treatment. Interestingly, we show that mouse p10 is able to induce cell cycle arrest and to protect against malignant transformation in vitro in a p53-dependent manner, suggesting a complex biological function for both the INK4A and INK4B loci in regulating the retinoblastoma and p53 pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids, primers, and probes. A genomic fragment containing the mouse INK4B locus (12) was used to study and characterize the mouse p10 sequence. This 6 kb fragment includes about 700 bp upstream of the p10- and p15-coding region and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). The mouse p10 cDNA was isolated by reverse transcription-PCR and subcloned into the pCR3.1 (Invitrogen) and pBabePuro vectors. Mouse p15 expression plasmids (pHM414, pBabePurop15) and the plasmid pMZN17 expressing mouse genomic N-Ras containing a codon 61 point mutation (N-RasT) were previously generated (12). An E1A12S expression vector (pB12-CMV-E1A12S) was kindly provided by Dr. Nevins (Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC). Plasmid p11.4 containing a mouse p53 cDNA insert driven by an SV40 promoter was obtained from J. Wallis and A. Levine (School of Natural Sciences, Institute for Advanced Study, Princeton, NJ). Mouse p19ARF cDNA was obtained by reverse transcription-PCR from wild-type mouse embryonic fibroblasts (MEF) total RNA and cloned into a pCR 3.1 vector (Invitrogen). Retroviral vectors for p21 and p27 expression were kindly provided by Dr. Mariano Barbacid (CNIO, Madrid, Spain).

The following primers were used in this work: oligo-dT-adapter (GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3') and adapter (5'-GACTCGAGTCGACATCG-3') primers were used in reverse transcription-PCR experiments; E2mp15F74 (5'-CTGCCACCCTTACCAGACCTGTGC-3'), E2mp15R257 (5'-TCTCCAGTGGCAGCGTGCAGATAC-3'), p15-20F (5'-GTCATGATGATGGGCAGCG-3') and p15E2R2 (5'-GCGTGCAGATACCTCGC-3') were p15-specific primers; E1mp10-F (5'-GTTGGGCGGCAGCAGT-3'), E2mp10-R (5'-CGCCCTCTGCCGGTAA-3'). ß-F (5'-GTGGGCCGCTCTAGGCACCAA-3'), mp10F1 (5'-GTAGCGGGGGTCCCCAGGCCTTACCG-3'), mp10E1R1 (5'-ACACAGCCAGCACCAGAGGGCTG-3') primers were used for p10-specific amplifications; mp15p2F was used for both p10 and p15 PCR assays; the primers for ß-actin were ß-R (5'-CTCTTTGATGTCACGCACGATTTC-3'), QmBactin-F (5'-TGTTACCAACTGGGACGACA-3') and QmBactin-R (5'-CTTTTCACGGTTGGCCTTAG-3'); primers for p21 were sense-p21 (5'-AGTGTGCCGTTGTCTCTTC-3') and antisense-p21 (5'-CCAGACGAAGTTGCCCTC-3'); finally, the primers for p27 were sense-p27 (5'-TCTCTTCGGCCCGGTCAATC-3') and antisense-p27 (5'-CTCTCCACCTCCTGCCACTC-3').

Specific probes for p10 and p15 were generated by amplifying exon 1ß and exon 2, respectively, with the primer pairs mp10F1/mp10E1R2 and p15-20F/p15E2R2, respectively. PCR products were run and purified twice before they were used in DNA and RNA hybridizations.

Expression studies by reverse transcription-PCR and Northern blotting. Total RNA was isolated from tissues and cells using Trizol (Life Technologies). To generate cDNA, 1 µg of total RNA was retrotranscribed using 0.4 µmol/L of an oligo-dT-adapter primer and SuperScript II RNase H-reverse transcriptase (Life Technologies) as described by the manufacturer. To determine the expression of p10, p15, and ß-actin in wild-type mouse tissues, the following pairs of primers were used: mp15p2F/adapter for p10 and p15 and ß-F/ß-R for ß-actin. Detection of ß-actin reverse transcription-PCR products was done by direct ethidium bromide staining of the 1.5% agarose gels, whereas detection of p10 and p15 bands was carried out by hybridization using p10- and p15-specific probes. To determine the expression of p10 in knock-out-p15 tissues, cDNAs from both knock-out-p15 and control wild-type tissues were used to coamplify ß-actin and p10 using the following pairs of primers: ß-F/ß-R for ß-actin and mp10F1/mp10E1R1 for p10. Detection of reverse transcription-PCR products was done by direct ethidium bromide staining.

Real time reverse transcription-PCR was done using primers for the mouse INK4B locus (E2mp15F74 and E2mp15R257 for p15, E1mp10-F and E2mp10-R for p10), p21 (sense-p21 and antisense-p21), p27 (sense-p27 and antisense-p27) and ß-actin (QmBactin-F and QmBactin-R). Quantitative PCR was done with the iCycler iQ System from Bio-Rad (Hercules, CA) using SYBR Green as DNA intercalator.

Cell culture, treatments, transfection, and retroviral infection assays. NIH-3T3 cells were obtained from the ATCC (CRL-1658) and maintained in DMEM (Life Technologies) supplemented with 10% calf serum and 1% penicillin G-streptomycin (Life Technologies). MEFs were isolated from C57BL/6J x DBA2 mice using E14.5 embryos as previously described (12). MEFs deficient for p15 and their wild-type littermates (13) were obtained from mice kindly provided by Latres et al. (13). All MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (Life Technologies), and 1% penicillin G-streptomycin (Life Technologies). For TGF-ß treatments, TGF-ß1 (Roche, Basel, Switzerland) was added to MEFs to a concentration of 100 pmol/L for the indicated times. Transfections were done using the calcium phosphate technique (14). Retroviral infections were done as previously reported (12). Briefly, three-passage MEFs or NIH-3T3 cells were infected with retroviral vectors carrying a puromycin resistance gene. Infected cell populations were selected for 5 days using puromycin at 2 µg/mL, and then, cell proliferation was analyzed by BrdUrd incorporation assays at the indicated times (see below).

Cell growth and malignant phenotype assays. For the growth curves, cells were seeded in triplicate in six-well plates (2.5 x 104 per well for the NIH-3T3 assays and 5 x 104 per well for the MEF curves) and counted daily during 7 days. Cellular proliferation was determined by BrdUrd incorporation assays. Cells were pulsed with 10 µmol/L BrdUrd, washed and fixed with 70% ethanol. Incorporated BrdUrd was detected with a FITC-conjugated anti-BrdUrd antibody (PharMingen, BD Biosciences, San Jose, CA). To determine DNA content by fluorescence-activated cell sorting, cells transduced with different retroviruses were harvested at the indicated times post-selection, washed, fixed with 70% ethanol, and resuspended in a solution containing propidium iodide at 50 mg/mL and DNase-free RNase at 0.5 mg/mL in PBS. Fluorescence was analyzed with a FACscan cytometer and data were interpreted using the CellQuest application (Becton Dickinson, Franklin Lakes, NJ).

For anchorage-independent growth studies (soft agar assays), MEFs were resuspended in 0.33% agar in DMEM supplemented with 10% fetal bovine serum at a density of 5 x 103/65 mm diameter plate and seeded onto solidified 0.5% agar–containing culture medium. Cultures were fed twice a week and colonies counted 2 weeks post-plating.

For the focus formation assays using MEFs, cells were cotransfected with 10 µg of pMZN17, 10 µg of E1A, and 10 µg of other expression plasmids. For the focus formation assays using NIH-3T3 fibroblasts, cells were cotransfected with 100 ng of pMZN17 and 5 µg of other expression plasmids. Two days post-transfection, cells were split (1:3 for MEFs and 1:5 for NIH-3T3 cells) and maintained in complete medium for 2 weeks. Foci were scored after staining with crystal violet.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the mouse p10. By analyzing a previously isolated p15 genomic sequence (12), we were able to determine that the organization of the mouse INK4B locus allows, as its human counterpart does, the generation of both p15 and p10 transcripts and proteins (Fig. 1A). The two variants derive from the alternative use of a splice donor site. As shown in Fig. 1B, the coding sequence of mouse p10 overlaps with the p15 coding sequence in all the sequences derived from exon 1, and has a unique 3' region that corresponds to the 5' end of the p15 intron 1. This p10-specific region has been termed exon 1ß in order to be consistent with the human p10 nomenclature. As a result of using the exon 1ß, a 67-amino acid protein is generated by the p10 transcript in comparison with the bigger protein (130 amino acids) encoded by the p15 coding sequence (Fig. 1C). Furthermore, the 22-amino acid, COOH terminus of the p10 protein is not present in p15. Regarding the protein structure, p10 has only the first of four ankyrin domains found in p15. It is interesting to note that mouse and human p10 proteins show a high degree of identity (77%, Fig. 1D).



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  		Figure 1. Characterization of the mouse p10 variant. A, schematic diagram of the INK4B locus showing the splicing pattern and the two coding sequences and proteins produced; B, nucleotide sequence alignment of the mouse coding sequences of p15 (top) and p10 (bottom). ATG is underlined whereas stop codons are indicated in boldface; C, amino acid sequence alignment of mouse p15 (top) and the predicted mouse p10 sequence (bottom); D, amino acid sequence comparison of human (top) and mouse (bottom) p10 proteins. Mouse and human p10-specific sequences are underlined, amino acids in boldface indicate the ankyrine domain.

 
Mouse tissues were examined for p15 and p10 expression by reverse transcription-PCR and subsequent hybridization with specific probes. As described in Materials and Methods, total RNAs from different mouse tissues were retrotranscribed using an oligo-dT-adapter primer to obtain cDNA from which both p10 and p15 were amplified by PCR using an adapter primer plus the forward primer mp15p2F, which is located in the 5'-untranslated region of the INK4B gene and it is common for the amplification of both gene products. PCR products were separated in agarose gels and blotted onto nitrocellulose membranes for subsequent hybridization with p10- and p15-specific probes. An expected band of ~1,300 bp was detected when a p15 probe was used, whereas a smaller one (~670 bp) was observed when membranes were hybridized with a p10-specific probe (Fig. 2A). To normalize, ß-actin expression was also determined in all samples. Like p15, p10 expression was detected in the majority of the analyzed mouse tissues. The highest p10 expression levels were detected in uterus and lung. Significant expression was shown in gut, kidney, and ovary, whereas very weak expression was found in spleen, thymus, and heart. No p10 expression was detected in brain, pancreas, and liver. Interestingly, the pattern of expression was very similar in all tissues for both p10 and p15 mRNAs. On the other hand, since the primers used in this reverse transcription-PCR experiment are common for p10 and p15, and given the fact that the p10-amplified fragment is smaller than the one amplified for p15, one should expect a preferential amplification of the former. However, we have found the opposite, a higher expression of p15 versus p10 in all tissues. Therefore, although this reverse transcription-PCR is not quantitative, the use of common primers and the subsequent hybridization with specific probes make it possible to suggest that p10 always showed lower expression levels than p15.



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  		Figure 2. p10 expression patterns in mouse tissues and MEFs. A, expression levels of p15 and p10 in wild-type mouse tissues (BR, brain; GT, gut; HR, heart; KD, kidney; LG, lung; LV, liver; OV, ovary; PC, pancreas; SP, spleen; TH, thymus; UT, uterus) were determined by reverse transcription-PCR and subsequent hybridization with specific probes as described in Materials and Methods. The ß-actin mRNA was measured as control; B, representative reverse transcription-PCR analysis of the expression of p10 in knock-out-p15 and wild-type tissues (TH, thymus; LG, lung; BR, brain; LV, liver; KD, kidney). Samples from the indicated tissues were used to coamplify ß-actin and p10 cDNAs. PCR products were separated in 1.5% agarose gels and visualized by staining with ethidium bromide; C, Northern blot showing p10 expression in knock-out-p15 and wild-type MEFs.

 
The fact that p10 is expressed at lower levels than p15 in all mouse tissues analyzed suggests a preferential expression of p15 versus p10. We used samples from knock-out-p15 mice (obtained by deletion of the second exon; ref. 13) and their wild-type littermates. We coamplified p10 and ß-actin as described in Materials and Methods. Even though similar ß-actin expression levels were detected in both wild-type and knock-out-p15 tissues, under these conditions, p10 bands could only be directly detected by ethidium bromide staining in knock-out-p15 samples (Fig. 2B). These results were confirmed when p15 and p10 mRNA levels were determined by Northern blot in MEFs derived from wild-type and knock-out-p15 mice. Using a p15 exon 1 probe that hybridized with both p15 and p10 transcripts, a small band corresponding to p10 transcripts was detected only in the knock-out-p15 MEFs, whereas wild-type MEFs showed a bigger band that corresponds to p15 mRNA (Fig. 2C).

Regulation of p15 and p10 by transforming growth factor-ß and oncogenic Ras. It is already known that p15 expression is up-regulated by TGF-ß (15). On the other hand, oncogenic Ras promotes the transcriptional induction of p15 (12). It has been shown that TGF-ß and oncogenic Ras activate the p15 promoter through three consensus Sp1 sites (12, 16). The genomic structure of the INK4B locus suggests that both p10 and p15 are under the transcriptional control of the same promoter. To test if p10 is regulated by TGF-ß and oncogenic Ras analogously to p15, real-time reverse transcription-PCR analysis of TGF-ß-treated and oncogenic-Ras-infected MEFs was carried out with p10- and p15-specific primers. As expected, a similar induction of both p10 and p15 expression was observed in TGF-ß-treated MEFs (Fig. 3A). Likewise, when MEFs were infected with a retroviral vector expressing an oncogenic form of N-Ras, p10 was induced as well as p15 (Fig. 3B). These results support the hypothesis that the expression of both p10 and p15 are under the same type of regulation.



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  		Figure 3. p10 expression is up-regulated by TGF-ß and oncogenic N-Ras in MEFs. A, MEFs were treated with TGF-ß at the indicated times and then p10 and p15 expression were measured by real-time reverse transcription-PCR. Values are indicated as fold change relative to the untreated MEFs; B, MEFs were infected with a control empty retrovirus or with retroviruses expressing oncogenic N-Ras. Positively infected cells were selected with puromycin. Five days post-selection, both p15 and p10 expression were determined by real time reverse transcription-PCR. Values are shown as fold change relative to the MEFs infected with an empty vector. In all cases, ß-actin expression was used to normalize the expression values. Columns, mean; bars, ± SE (n = 3).

 
Mouse p10 induces cell cycle arrest and inhibits malignant transformation in vitro. Since human p10 negatively regulates cellular growth, we decided to study the role of mouse p10 in cell cycle regulation of both primary and immortalized mouse fibroblasts. Due to the low efficiencies of conventional gene transfer methods in primary fibroblasts and the limited life span of this type of cells, we chose retrovirus-mediated gene-transfer for all the growth-regulation experiments. We followed the time frame and protocol previously described (12).

First, we tested the effect of p10 expression in the cell cycle regulation of a line of immortalized mouse fibroblasts, NIH-3T3, in which we verified that the INK4B locus is completely lost (data not shown). We transduced NIH-3T3 fibroblasts with retroviral vectors carrying a puromycin resistance gene together with either p15 or p10. An empty vector and a p21 expressing vector were transduced as a negative control and a CDK4/CDK6-independent cell cycle negative regulator, respectively. After 5 days of selection with puromycin, growth rate was analyzed. Although p15-infected NIH-3T3 cells grew slower than those infected with the empty vector (Fig. 4A), at day 4 post-seeding, the inhibitory effect of p10 was weaker than that of p15 (data not shown) and indistinguishable from cells infected with an empty vector at day 7 post-seeding (Fig. 4A).



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  		Figure 4. Mouse p10 induces cell cycle arrest in mouse primary and immortalized fibroblasts. A, NIH-3T3 cells were infected with pBabePuro-empty, pBabePuro-p10, pBabePuro-p21, and pBabePuro-p15 retroviruses. After selection with puromycin, a cell growth assay was done. Cells were counted daily for 7 days; B, wild-type MEFs were infected with pBabePuro-empty, pBabePuro-p10, pBabePuro-p27, and pBabePuro-p15 retroviruses and assayed for cell growth as described in Materials and Methods. Expression of each retroviral construct (in A and B) was determined by real-time reverse transcription-PCR. Data for these representative experiments are as follows: 1.83 ± 0.61 for p15; 1.84 ± 0.45 for p10, 1.79 ± 0.22 for p21 (A); 2.94 ± 0.08 for p15, 2.96 ± 0.47 for p10, and 1.39 ± 0.17 for p27 (B). Values were normalized with ß-actin expression and shown as fold change relative to the cells infected with an empty vector; C, MEF proliferation was quantified by BrdUrd incorporation. Column, mean; bars, ± SE (n = 3), percentage of BrdUrd-positive cells at days 2 and 4 post-selection in MEFs infected with pBabePuro-empty (black columns), pBabePuro-p10 (white columns), and pBabePuro-p15 (gray columns) retroviruses; D, representative experiment from (C) showing a propidium iodide staining for DNA content. Black, gray, white, and light gray correspond to wild-type MEFs infected with empty, p15, p10, and p27 retroviruses, respectively. Columns, mean; bars, ± SE.

 
To further analyze the effect of p10 in mouse fibroblasts, we carried out the same growth curve assay with retrovirally infected MEFs. In this case, mouse p10 showed a strong cell cycle inhibition phenotype when ectopically expressed in MEFs compared with immortalized cells (Fig. 4B). Indeed, a clear inhibitory effect in cell cycle proliferation was observed in wild-type MEFs infected with p10- or p15-expressing retroviruses (Fig. 4C and D). To quantify the effect of p10 in MEFs, we did a BrdUrd incorporation assay and a propidium iodide staining for DNA content as described in Materials and Methods. As shown in Fig. 4C, retroviral expression of p10 in MEFs induces a significant decrease in the number of cells that progress to S phase. Interestingly, this cell cycle arrest induced by p10 was not different from the one induced by p15. To determine how p10 was able to block cell cycle progression, we decided to test different options including G1 or G2-M arrest and cell death. When MEFs were studied for DNA content, p10 induced in primary fibroblasts an accumulation of cells in G1, whereas no differences were found for the number of cells in G2-M (Fig. 4D). This cell cycle pattern is very similar to the one induced by p15 or p27 overexpression characterized by an increased percentage of cells in G1 together with a decrease in the number of cells in S phase, and without significant changes in any other category or phase. With respect to the induction of apoptosis, no significant differences were found between p10 and empty vector–infected cells for the sub-G0 population (Fig. 4D). Likewise, when cells were stained with Annexin V to determine the number of apoptotic cells, no significant differences were found between MEFs infected with an empty vector or a p10 expression retrovirus (data not shown). Therefore, the cell cycle inhibitory effect induced by p10 is based in a G1 arrest mechanism.

It has already been shown that p15 is induced as a response to oncogenic signals (12) and is inactivated in some tumor types (17–19). In accordance with these results, we have also shown that p15-deficient MEFs were more susceptible to transformation by oncogenic N-Ras than wild-type MEFs (12). Therefore, p15 has clear antimalignant properties both in vivo and in vitro. To test the possibility that p10 shares some of these properties with p15 in vitro we did a foci formation assay in wild-type MEFs. Indeed, as shown in Fig. 5A, p10 inhibited foci formation in MEFs transformed with oncogenic N-Ras plus E1A. A similar inhibitory effect was observed for the anchorage-independent phenotype induced by both N-Ras plus E1A in wild-type MEFs (Fig. 5B). In both assays, the inhibitory effect of the ectopic expression of p10 was similar to the one induced by p15 overexpression.



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  		Figure 5. Mouse p10 protects from N-RasT-induced malignant phenotype. Transformation assays (A) and anchorage-independent cell growth assays (B) were done in wild-type MEFs. Cells were cotransfected with the indicated expression vectors as described in Materials and Methods. Two weeks later, foci and colonies were counted. Columns, number of foci or colonies as a percentage of the number found among those MEFs cotransfected with E1A plus N-RasT alone. Columns, mean; bars, ± SE (n = 3).

 
The p53 pathway is necessary for p10 function. Because of the differential p10-induced response that we found in NIH-3T3 cells versus MEFs (Fig. 4), and since NIH-3T3 cells have a defective p53 pathway—by the loss of ARF (20)—we decided to test the effect of p10 on MEFs defective in the p53 pathway. Retroviruses carrying the puromycin resistance gene either empty or containing p10 or p15 were used to infect p53–/– MEFs. After the period of antibiotic selection, cell cycle proliferation was analyzed using a BrdUrd-incorporation assay. At day 2 post-selection, p53–/– MEFs infected with a p15-expressing vector showed a weak inhibition (<20%) of BrdUrd incorporation when compared with wild-type MEFs (30-20%, Fig. 4C). More importantly, this inhibitory effect completely disappeared at day 4 post-selection in p53–/– MEFs (Fig. 6A), whereas it is still present in wild-type MEFs (Fig. 4C). In the case of the p53–/– MEFs infected with p10, we could not detect any significant differences on BrdUrd-incorporation in comparison with the cells infected with an empty vector (Fig. 6A), whereas for the wild-type MEFs infected with the same retroviral vector, we showed a clear inhibitory effect (40-50% decrease in the percentage of BrdUrd-positive cells, Fig. 4C) To confirm the fact that p10 (and p15) are not able to induce cell cycle arrest in p53–/– MEFs, we did a cell growth curve assay using empty vector, p10-, p15-, and p27-infected cells. As shown in Fig. 6B, only the positive control, p27, induced a clear growth inhibition, whereas both p10- and p15-infected p53–/– MEFs behaved similar to those infected with an empty retroviral vector.



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  		Figure 6. p53 is necessary for p10 function. A, proliferation of p53–/– MEFs infected with the control, p10, and p15 retroviral vectors as described in Materials and Methods. Columns show the percentage of BrdUrd-positive cells, mean ± SE (n = 3), at days 2 and 4 post-selection; B, cell growth curves for p53–/– MEFs infected with pBabePuro-empty, pBabePuro-p10, pBabePuro-p27, and pBabePuro-p15 retroviruses as described in Materials and Methods. Results, mean ± SE, are representative of three separate experiments. Expression of each retroviral construct was determined by real-time reverse transcription-PCR as in Fig. 4A and B. Data for this representative experiment are as follows: 2.51 ± 0.19 for p15, 2.67 ± 0.25 for p10, and 1.46 ± 0.04 for p27; C, transformation assay done in NIH-3T3 cells as described in Materials and Methods. Cells were cotransfected with N-RasT plus either control, p15, or p10 vectors; D, transformation assay done in NIH-3T3 cells cotransfected with N-RasT plus p53 and either control or p10 vectors. Columns, number of foci as a percentage of the number found among those NIH-3T3 cells cotransfected with p53 plus N-RasT and control vector. Columns, mean; bars, ± SE (n = 3).

 
In a second set of experiments, we tested whether or not the antitransforming properties of p10 were p53-dependent. First, we did a focus formation assay with NIH-3T3 cells, which, as it was mentioned above, have the p53 function impaired. As it was previously reported, p15 significantly reduced the number of foci induced by oncogenic N-Ras. However, ectopic expression of p10 had no effect in the transforming phenotype induced by N-Ras (Fig. 6C). We repeated the foci formation assay in NIH-3T3 cells cotransfected with p53. Importantly, in NIH-3T3 cells, p10 cooperated with p53 in the inhibition of N-RasT-induced foci formation (Fig. 6D). Taken together, all these data suggest that both the cell cycle arrest and the antimalignant effect induced by p10 are p53-dependent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the last two decades, many molecules have been associated with the regulation of cellular proliferation. Nowadays, we know that a complex molecular network regulates cell cycle progression in a fine-tuned manner. The INK4 proteins constitute one of the groups of cell cycle regulators with inhibitory properties. The INK4 proteins induce cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by CDK4-6/cyclin-D complexes. The INK4A gene, the best known of all the INK4 family members, is involved in the development and progression of different tumor types (21). Interestingly, three different transcripts are coded by this locus. In addition to the p16INK4A and ARF transcripts, which encode for two different proteins through the use of unique first exons and alternative reading frames, another variant, termed p12, has been described (10). In this case, the 12 kDa protein is encoded by a shorter transcript that shares the reading frame with the p16INK4A mRNA. The INK4B gene has also been associated with tumorigenesis, especially with malignant lymphoproliferative disorders (18, 19, 22). Like the INK4A locus, different transcripts are synthesized by this locus. In addition to the originally described p15 transcript (15), an alternative spliced form, p10, has been reported in humans (11).

In this work, we have characterized the mouse p10 splicing variant and we have studied some of its functional properties. From a structural point of view, mouse p10 is almost identical to its human orthologue. Both p10 variants use the same reading frame as p15 and share with it the first exon. A p10-specific region, termed exon 1ß, rises from the read-through of a donor splicing site at the end of p15 exon 1. Thus, intronic sequences for p15 are going to constitute exon 1ß for p10. Therefore, p10 transcripts produce a protein composed of the INK4B first exon and a novel intron-derived COOH terminus. Both human and mouse p10 amino acid sequences have a high degree of identity (77%). They both share an ankyrin domain with p15 and each has a p10-specific COOH-terminal region, the function of which is unknown. At the functional level, mouse p10 shows some of the cell cycle inhibitory properties that have been described for human p10 (11). First, stable expression of either human or mouse proteins do not have a significant effect on the growth of NIH-3T3 cells (ref. 11; Fig. 4A). Second, we found that, like human p10, mouse p10 expression is induced by TGF-ß. We have previously shown that p15 expression is up-regulated by oncogenic insults (12). In this work, we show that the oncogenic form of N-Ras up-regulates mouse p10 expression in a very similar way (Fig. 3B). Third, we have detected p10 expression by reverse transcription-PCR in the majority of mouse normal tissues. Human p10 expression has only been analyzed in cell lines (11). Here, we detect a broad expression pattern in mouse tissues. Interestingly, although mouse p10 expression was weaker than that found for mouse p15 in all analyzed tissues, a similar pattern of expression was found for both INK4B variants in those tissues. These results, together with the fact that both mouse p15 and p10 expression are up-regulated by TGF-ß and oncogenic N-Ras, suggest that both transcripts are regulated in a similar way.

In addition to the structural and functional similarities between mouse and human p10, the present work describes several new features of p10 function, particularly those related with its inhibitory and antimalignant properties. Although the effects of both human and mouse p10 variants on cell cycle progression have been studied in immortalized cell lines (ref. 11; Fig. 4A) no study has been previously done in primary cells. Here we have shown that, whereas p10 does not have any inhibitory role in immortalized cells, it induces a clear cell cycle inhibitory effect on primary cells. This result suggests that some molecules and/or pathways that are necessary for p10 function are not present in immortalized cell lines. The loss of the INK4A locus is a common event in the immortalization of primary cell lines (20, 23–26). This genetic alteration affects the p53 and retinoblastoma pathways since both p16 and ARF proteins are frequently lost. It has been previously shown that human p10 does not bind CDK4 and CDK6 proteins. Furthermore, p12, a splicing variant similar to p10 that is encoded by the INK4A locus, is able to suppress growth in a retinoblastoma-independent manner. Therefore, the main candidates to be involved in the p10-dependent inhibition in primary fibroblasts are the members of the p53 pathway. Indeed, in this work, we have shown that p53 is necessary for the p10-induced cell cycle arrest in primary fibroblasts (Fig. 6A and B). However, the mechanism(s) by which p10 proteins regulate or are regulated by the p53 pathway are still unknown.

Another interesting new finding is the antimalignant properties of mouse p10. ARF, p16, and p15 proteins have clear antimalignant transformation properties in vivo and in vitro (27, 28). However, although human p10 and its INK4A equivalent, p12, are able to inhibit cell cycle progression, no information exists about their effectiveness in reversing the malignant phenotype. Importantly, we have shown that mouse p10 is able to inhibit N-RasT plus E1A-induced foci formation and anchorage-independent growth in wild-type MEFS. Moreover, as we have shown in foci formation assays done in NIH-3T3 cells, the antitransforming properties of mouse p10 are p53-dependent. These results have important implications in the low (<10%) tumor susceptibility phenotype of p15-null mice (13). Since p15-null cells express (or even overexpress) p10 (Fig. 2B and C), it is possible that p15-defective cells and mice are defective in the retinoblastoma-dependent inhibitory properties of the INK4B locus, but display normal (or even higher) p53-dependent tumor suppression properties conferred by p10.

A number of critical differences have been described between p15 and p10. First, the p10 protein only contains the first of the four ankyrin p15 repeats. It is already known that the second and third ankyrin repeats of p16 are necessary to interact with CDK6 (29). In addition, human p10 does not bind to either CDK4 or CDK6 (11). Therefore, the mechanism(s) by which p10 inhibits cell growth must be CDK4/6-independent. Interestingly, it has been shown that p15 is able to inhibit cell growth in a retinoblastoma-independent fashion (30). Here, our data suggest that the inhibitory properties of p15 are p53-dependent (Fig. 6A and B). Therefore, both p10 and p15 share the p53-pathway as the cell cycle regulatory system to inhibit cell proliferation. However, both INK4B products differ in their antimalignant properties since, as we have shown in this work, although p15 is able to inhibit cell transformation in cells negative for p53-function (NIH-3T3), this is not the case of p10 (Fig. 6C).

What is the relevance of p10 expression and function? One of the most interesting conclusions derived from this work is the fact that the INK4B locus, like the INK4A locus, expresses transcripts that are involved in the p53 and retinoblastoma protein pathways, and subsequently, cell cycle progression and tumorigenesis. Herein, p15 and p16 products act mainly via retinoblastoma, whereas p19/ARF, p10 and, to a lesser extent, p15 functions are p53-dependent. Interestingly, both loci are located at the same chromosomal region in the human and mouse genome, a hotspot for cancer-associated deletions (18, 31). More importantly, several specific genetic alterations within these loci have been associated with the development and progression of different tumors (27). Whereas specific inactivating point mutations have been described in either p16 or p19ARF and can discriminate their role in tumor development in specific samples (21), cancer-associated point mutations are rare in the INK4B locus (18, 32). Instead, INK4B is inactivated in human and murine cancer by deletion or promoter hypermethylation (17–19, 33). Since both p15 and p10 use the same promoter, each of these events would prevent the expression of both p15 and p10 in tumor cells. Interestingly, p15-deficient mice still express p10 (Fig. 2C) and could, therefore, only partially inactivate the function of the INK4B locus, opening the door for revisiting the true tumor suppressor properties of this gene. The present study also provides the bases for the identification of molecules associated with p10, which act through the p53-pathway, as potential targets for future anticancer therapies.


   Acknowledgments
 
Grant support: NIH grant CA36327 (A. Pellicer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Mariano Barbacid (CNIO, Spain) for providing the knock-out-p15 mice, Karoline Asbell for her technical assistance, John Hirst (New York University) for his help with flow cytometric analysis, and Christine Pampeno for critical reading of the manuscript. I. Pérez de Castro was a recipient of a postdoctoral fellowship and is a recipient of a Ramón y Cajal contract from the Ministry of Education and Science (Madrid, Spain).

Received 11/ 5/03. Revised 1/17/05. Accepted 2/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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