Increased gene dosage of Ink4a/Arf results in cancer resistance and normal aging

Abstract

Mammalian genes frequently present allelic variants that differ in their expression levels and that, in the case of tumor suppressor genes, can be of relevance for cancer susceptibility and aging. We report here the characterization of a novel mouse model with increased activity for the Ink4a and Arf tumor suppressors. We have generated a "super Ink4a/Arf" mouse strain carrying a transgenic copy of the entire Ink4a/Arf locus. Cells derived from super Ink4a/Arf mice have increased resistance to in vitro immortalization and oncogenic transformation. Importantly, super Ink4a/Arf mice manifest higher resistance to cancer compared to normal, nontransgenic, mice. Finally, super Ink4a/Arf mice have normal aging and lifespan. Together, these results indicate that modest increases in the activity of the Ink4a/Arf tumor suppressor result in a beneficial cancer-resistant phenotype without affecting normal viability or aging.

Figure 1.

Generation of a transgenic Ink4a/Arf allele. (A) Structure of the transgene. The transgene contains the complete exon structure of three genes, Ink4b, Arf, and Ink4a. Boxes labeled SP6 and T7 represent short terminal sequences (of 20 bp) used for the identification of the transgene by PCR (see Materials and Methods). A thick bar near the SP6 terminus indicates the location of the probe used for the Southern blot shown in C. (B) Fluorescent in situ hybridization of the transgene in a representative MEF metaphase. The fluorescent probe consists of the complete transgene. The transgene is located in a chromosome that is smaller than the two homologs containing the endogenous alleles. (C) Southern blot to detect the SP6-terminal region of the transgene. Genomic DNA digested with BamHI produced a band of ∼4 kb, present in both wild-type and transgenic mice, and a single additional band of ∼6 kb specific to transgenic mice. The probe used consisted of a PCR fragment of ∼380 bp (schematically shown in A) that was generated by primers SP6-PAC and SP6-tg (see Materials and Methods). (D) PCR genotyping of the various Ink4a/Arf alleles. (Top) Detection of the transgene by a PCR reaction based on the T7-terminal sequence of the transgene. (Bottom) The endogenous Ink4a/Arf allele was indirectly genotyped through a linked polymorphism in the Tyrp1 (tyrosinase-related protein 1) locus, which is located at a genetic distance of ∼4.7 cM. The Ink4a/Arf-wild-type (wt) allele is linked to the wild-type Tyrp1 allele, whereas the Ink4a/Arf-null allele is linked to the mutant Tyrp1(brown) allele (Sviderskaya et al. 2003). The two Tyrp1 alleles can be distinguished by a diagnostic TaqI site (Zdarsky et al. 1990) present in Tyrp1-wt (linked to Ink4a/Arf-wt), but absent in Tyrp1(brown) (linked to Ink4a/Arf-null). A PCR product that contains the reported TaqI polymorphism was digested with TaqI to determine the presence or absence of the TaqI site.

Figure 2.

Function of the Ink4a/Arf transgene in MEFs. (A) Activation of the transgenic Ink4a/Arf allele in response to in vitro culture stress. MEFs of the indicated genotype were serially passaged according to the 3T3 protocol. In the case of Ink4a/Arf (–/–;tg/·) MEFs (right panels), transgene-produced p16^Ink4a and p19^Arf reached maximal levels at around passage 35. In the case of Ink4a/Arf (+/–) MEFs (left panels), maximal levels were reached at around passage 4. In both cases, cells expressing p16^Ink4a and p19^Arf, either from the transgene or from the endogenous allele, were overgrown by cells that had lost expression of p16^Ink4a and p19^Arf. This overall pattern was reproducibly observed in four independent (–/–;tg/·) MEF cultures, each from a different embryo (see Supplementary Fig. S1). (B) Activation of the transgenic Ink4a/Arf allele in response to Ras-induced oncogenic stress. MEFs of the indicated genotype and passage were retrovirally transduced with an empty vector or with a vector expressing oncogenic H-RasV12, and the levels of p16^Ink4a and p19^Arf were determined at day 2 post-selection (see Supplemental Material). Immunoblots are representative of three assays with independent MEF cultures. (C) The transgenic Ink4a/Arf allele provides neoplastic resistance to oncogenic Ras. (Black bars) MEFs of the indicated genotype and passage were infected with oncogenic Ras or with empty vector, and their proliferation was measured at day 0 and day 4 post-selection. The ratio between day 4 and day 0 in Ras-infected cells was normalized as the percentage of the corresponding ratio in vector-infected cells. (Gray bars) MEFs (10⁶ cells) were transfected with a plasmid encoding oncogenic Ras, and 3 wk later the total number of neoplastic foci was scored. Data for proliferation and neoplastic foci correspond to the average and standard deviation of three and five assays, respectively, with independent MEF cultures. (D) Activation of the transgenic Ink4a/Arf allele in response to E1a-induced oncogenic stress. MEFs were retrovirally transduced with an empty vector or with a vector expressing E1a, and the levels of p16^Ink4a and p19^Arf were determined by immunoblotting. Data are representative of three assays with independent MEF cultures. (E) The transgenic Ink4a/Arf allele provides increased sensitivity to E1a-mediated apoptosis. MEFs retrovirally transduced with E1a (+) or empty vector (–) were treated or not with doxorubicin, as indicated, and apoptotic cells were quantified 24 h later by cytometry. Data correspond to a representative assay from a total of three assays with independent MEF cultures.

Figure 3.

Function of the Ink4a/Arf transgene in vivo. (A) The transgenic Ink4a/Arf allele rescues the aberrant eye phenotype of Arf-deficient mice. The pictures show representative sections of eyes from adult mice (8–12 mo old) of the indicated genotypes stained with hematoxylin and eosin. In agreement with the previously reported phenotype of Arf-null mice (McKeller et al. 2002), the eyes of Ink4a/Arf (–/–) mice are severely distorted due to an aberrant layer of tissue derived from the embryonic hyaline vasculature (marked with an asterisk). The eyes of Ink4a/Arf (–/–;tg/·) mice were normal and indistinguishable from the eyes of Ink4a/Arf (+/–) mice. (B) The transgenic Ink4a/Arf allele directs the expression of Ink4a-specific and Arf-specific mRNA. Ink4a and Arf transcripts were detected by real-time quantitative RT–PCR using primers that specifically distinguish both transcripts. Values indicate the average and standard deviation of the difference in PCR cycles (ΔCt) with respect to β-actin (it should be noted that the higher the value, the lower the expression). The observed differences were not statistically significant. Total RNA was obtained from testis of mice (∼1.5 yr old) of the indicated genotype. Each measure is derived from at least two independent assays, each of them with triplicate PCRs. (C) Transgenic Ink4a/Arf mice express transgene-derived p16^Ink4a and p19^Arf proteins. Protein extracts were obtained from spleen and testis of mice (∼1.5 yr old) of the indicated genotype. The samples from testis in the Ink4a and Arf immunoblots correspond to the same mice. The two lanes labeled (–/–;tg/·) correspond to different mice. (D) The transgenic Ink4a/Arf allele delays spontaneous tumorigenesis in the absence of endogenous Ink4a/Arf alleles. Survival curve of mice of the indicated genotype. The size of each group of mice was as follows: (–/–), n = 32; (+/–), n = 14; (–/–;tg/·), n = 36. Table 1 shows the incidence of spontaneous neoplasias in these mice. (E) The transgenic Ink4a/Arf allele delays DMBA-induced tumorigenesis in the absence of endogenous Ink4a/Arf alleles. Survival curve of mice treated with DMBA to induce lymphomas and fibrosarcomas (see Table 1). The size of each group of mice was as follows: (–/–), n = 14; (+/–), n = 14; (–/–;tg/·), n = 13. (F) The transgenic Ink4a/Arf allele delays pristane-induced tumorigenesis in the absence of endogenous Ink4a/Arf alleles. Survival curve of mice treated with pristane to induce plasmacytomas and peritoneal tumors. The size of each group of mice was as follows: (–/–), n = 13; (+/–), n = 7; and (–/–;tg/·), n = 14.

Figure 4.

MEFs derived from super Ink4a/Arf mice have increased resistance to immortalization and oncogenic transformation. (A) Super Ink4a/Arf MEFs have increased resistance to spontaneous immortalization. Cultures derived from independent embryos, either wild-type (n = 27) or super Ink4a/Arf (n = 7) were passaged according to the 3T3 protocol. Cultures were maintained until immortalization. Bars represent the percentage of cultures that have attained immortalization at each of the indicated passage intervals. (B) Spontaneously immortalized super Ink4a/Arf MEFs preferentially lose p53 function. Spontaneously immortalized MEFs derived from wild-type (n = 26) or super Ink4a/Arf (n = 7) MEFs (see A) were analyzed for the loss of p19^Arf (by immunoblot) and for the loss of p53 function (by testing the stabilization of p53 upon doxorubicin treatment). Bars represent the percentage of analyzed cultures according to their mechanism of immortalization. (C) Super Ink4a/Arf MEFs have increased sensitivity to E1a-mediated apoptosis. Early-passage MEFs of the indicated genotype were retrovirally transduced with E1a (+) or empty vector (–), and the basal level of apoptosis was measured by cytometry. Data correspond to the average and standard deviation of four assays with independent MEF cultures. (D) Super Ink4a/Arf MEFs present increased levels of p19^Arf in response to E1a. MEFs from the experiment shown in C were used to prepare protein extracts, and the levels of p19^Arf were measured by immunoblots. Similar results were obtained in two additional independent assays. (E) Super Ink4a/Arf MEFs have increased resistance to neoplastic transformation by cooperating oncogenes Ras and E1a. Early-passage MEFs of the indicated genotype were retrovirally transduced with a construct that simultaneously expresses oncogenic Ras and E1a. After selection, infected MEFs (2 × 10⁵ cells) were plated and, after 3 wk, the number of neoplastic colonies was scored. Data correspond to the average and standard deviation of three assays with independent MEF cultures.

Figure 5.

Super Ink4a/Arf mice have increased resistance to cancer. (A) Moderately increased expression of p16^Ink4a, p19^Arf, and p15^Ink4b in super Ink4a/Arf mice. Extracts from the indicated organs were obtained from mice that were ∼1.5 yr old. In some tissues, expression levels were too low and, therefore, the corresponding immunoblots are not shown. For each tissue, similar data were obtained with at least two mice of each genotype. (B) Super Ink4a/Arf mice have increased resistance to DMBA/TPA-induced skin papillomas. The figure shows the average number of papillomas at every week, and their relative size distribution. Initiation treatments (DMBA application) were applied when mice were 1 wk old and, 3 wk later, promotion treatments began (TPA applications) and continued for an additional 12 wk. At 16 wk of age (indicated by ^*) treatment was terminated. The size of each group of mice was as follows: (+/+), n = 15; (+/+;tg/·), n = 7. (C) Super Ink4a/Arf mice have increased resistance to 3MC-induced fibrosarcomas. Survival curves of mice treated with 3MC. The size of each group of mice was as follows: (+/+), n = 8; (+/+;tg/·), n = 7. (D) Super Ink4a/Arf mice have increased resistance to pristane-induced plasmacytomas. Survival curves of mice treated with pristane. The size of each group of mice was as follows: (+/+), n = 16; (+/+;tg/·), n = 10 (E) Super Ink4a/Arf mice have increased resistance to urethane-induced lung adenomas. Dots indicate the number of visible tumors per mouse; horizontal lines indicate the average. The size of each group of mice was as follows: (+/+), n = 4; (+/+;tg/·), n = 6.

Figure 6.

Normal viability and aging of super Ink4a/Arf mice. (A) Super Ink4a/Arf mice have normal fertility. Average litter size and standard deviation of the indicated crosses. The number of litters scored for each type of cross is indicated (n). (B) Super Ink4a/Arf mice have normal lifespan. Cohorts of wild-type (n = 22) and super Ink4a/Arf (n = 22) littermate mice were followed for a period of 27 mo. The figure shows a Kaplan-Meier representation of the survival of the two groups of mice. (C) Old super Ink4a/Arf mice have normal weight. Average weight and standard deviation of the indicated mice. All mice were 2 yr of age. The size of each group of mice was as follows: (+/+) females, n = 5; (+/+) males, n = 8; (+/+;tg/·) females, n = 7; (+/+;tg/·) males, n = 9. (D) Similar lordokyphosis in old super Ink4a/Arf and wild-type mice. Dots indicate the angle of the dorsal spine measured from X-radiographs of individual mice at 2 yr of age, as described (Garcia-Cao et al. 2002). Short lines indicate the average. The size of each group of mice was as follows: (+/+), n = 3; (+/+;tg/·), n = 3.