GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

p16INK4A and p19ARF act in overlapping pathways in cellular immortalization

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.     

Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells

Somatic cells can be reset to oncogene-induced senescent (OIS) cells or induced pluripotent stem (iPS) cells by expressing specified factors. The INK4/ARF locus encodes p15(INK4b) , ARF, and p16(INK4a) genes in human chromosome 9p21, the products of which are known as common key reprogramming regulators. Compared with growing fibroblasts, the CCCTC-binding factor CTCF is remarkably up-regulated in iPS cells with silencing of the three genes in the locus and is reversely down-regulated in OIS cells with high expression of p15(INK4b) and p16(INK4a) genes. There are at least three CTCF-enriched sites in the INK4/ARF locus, which possess chromatin loop-forming activities. These CTCF-enriched sites and the p16(INK4a) promoter associate to form compact chromatin loops in growing fibroblasts, while CTCF depletion disrupts the loop structure. Interestingly, the loose chromatin structure is found in OIS cells. In addition, the INK4/ARF locus has an intermediate type of chromatin compaction in iPS cells. These results suggest that senescent cells have distinct higher-order chromatin signature in the INK4/ARF locus.     

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.     

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b)

The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.     

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.     

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.     

Reduced expression of INK4a/ARF genes in stem-like sphere cells from rat sarcomas

The presence of cancer stem cells, in both hematopoietic and solid malignancies, has been recently linked to their pathogenesis. We aimed to identify the characteristics and stem-like properties of sphere-colony forming cells in rat osteosarcoma and malignant fibrous histiocytoma cell lines. The results showed that both cell lines possessed an ability to form spherical, clonally expanding colonies in anchorage-independent, serum-starved conditions in N2/1% methylcellulose medium. The sphere cells showed stem-like properties with the ability to self-renew, and expressed the stem cell-related STAT3 and Bmi1 genes. Interestingly, spheres from both sarcomas remarkably decreased the expression of INK4a/ARF locus genes, p16(INK4a) and p19(ARF), which could be related to the resistance against cell senescence and apoptosis. Spheres showed strong tumorigenicity with metastatic potential in vivo via the inoculation into syngeneic rats, suggesting the presence of these populations might contribute to the tumor development such as metastasis via the resistance to apoptotic stimuli.