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bmi-1 regulates proliferation and senescencecell-through the ink4a locus


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matergic receptor may subserve behavioural responses to noxious heat and cold. Although glutamatergic synapses mediate pain sensation at all intensities, recent studies26,27 indicate that substance P and/or neurokinin A are important in sensing intense pain. Thus, it appears that both intensity and modality of pain are coded by different subtypes of postsynaptic receptor and by the release of different transmitters from primary afferent ?bres. M
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Methods

Spinal slices. Whole-cell recordings were made from spinal slices of rats at

postnatal days 4±21 as described28. EPSCs were evoked at 0.05 Hz with a bipolar tungsten electrode (stimulus width 0.1 or 0.4 ms) placed at the DREZ or dorsal root nerve. Monosynaptic EPSCs were identi?ed as described28. Only monosynaptic EPSCs were studied. Currents were ?ltered at 1 kHz and digitized at 5 kHz. Bicuculline methiodide (10 mM) and strychnine hydrochloride (1 mM) were added to the perfusion solution. Statistical comparisons were made using one-way analyses of variance (ANOVAs; Dunnett test for post-hoc comparison) or Student's t-test. P , 0:05 was considered to be signi?cant. SYM 2206 and SYM 2081 were from Tocris±Cookson; other compounds were from RBI or Sigma. Cultured neurons. Neurons were dissociated from the dorsal half of spinal cord slices and maintained for 7±14 days in culture using standard methods15. Whole-cell pipettes were ?lled with (in mM): 140 caesium glucuronate, 10 EGTA, 10 HEPES, 5 CsCl, 5 MgCl2, 5 ATP and 1 GTP, pH 7.4. Drugs were dissolved in (in mM) 160 NaCl, 10 HEPES, 2 CaCl2 plus 500 nM tetrodotoxin (pH 7.4), and applied by rapid local perfusion from a multibarrelled pipette as described15. Labelling with ?uorescent dye. Rats were anaesthetized with halothane (2± 3%) delivered through a nose cone (with 30% O2 balanced with N2) and were positioned in a stereotaxic apparatus. Fluorescent tracer (0.5±1 ml DiI, 0.2% in dimethylsulphoxide, or rhodamine latex microspheres) was injected into one side of the lateral and medial part of the thalamus24. Two days after injection, the somata of ascending projection cells were visualized under epi?uorescent illumination with a rhodamine ?lter set. Behavioural tests. The tail-?ick re?ex and hot-plate (50 8C) and cold-plate (0 8C) tests were measured as described25. Cold stimuli (0 8C) are believed to be noxious and to activate speci?c cold nociceptors25. During intrathecal injection, mice or rats were anaesthetized with halothane (2%). Injections of drugs (mice, 5 ml; rats, 15 ml) were made with the steel tip of a 30-gauge needle connected by a 1-ft length of ?exible PE-10 tubing to a 50-ml syringe. Saline was used as a control. After the injection, it took 2±3 min for animals to recover. Data are presented as maximum possible inhibition ?MPI? ? ?response latency 2 baseline response latency?=?cutoff time 2 baseline response latency? 3 100.
Received 22 October; accepted 23 November 1998. 1. Bliss, T. V. P. & Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31±39 (1993). 2. Lerma, J., Morales, M., Vicente, M. A. & Herreras, O. Glutamate receptors of the kainate type and synaptic transmission. Trends Neurosci. 20, 9±12 (1997). 3. Castillo, P. E., Malenka, R. C. & Nicoll, R. A. Kainate receptors mediate a slow synaptic current in hippocampal CA3 neurons. Nature 388, 182±186 (1997). 4. Vignes, M. & Collingridge, G. L. The synaptic activation of kainate receptors. Nature 388, 179±182 (1997). 5. Mulle, C. et al. Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-de?cient mice. Nature 392, 601±605 (1998). 6. Yaksh, T. L. & Malmberg, A. B. in Textbook of Pain (edited by Wall, P. D. & Melzack, R.) 165±200 (Churchill Livingstone, New York, 1994). 7. Yoshimura, M. & Jessell, T. M. Amino acid-mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord. J. Physiol. (Lond.) 430, 315±335 (1990). 8. Kumazawa, T. & Perl, E. R. Excitation of marginal and substantia gelatinosa neurons in the primate spinal cord: indications of their place in dorsal horn function organization. J. Comp. Neurol. 177, 417± 434 (1978). 9. Light, A. R., Trevino, D. L. & Perl, E. R. Morphological features of functionally de?ned neurons in the marginal zone and substantia gelatinosa of the spinal dorsal horn. J. Comp. Neurol. 186, 151±171 (1979). 10. Bleakman, D. et al. Activity of 2,3-benzodiazepines at naive rat and recombinant human glutamate receptors in vitro; stereospeci?city and selectivity pro?les. Neuropharmacol. 35, 1689±1702 (1996). 11. Pelletier, J. C., Hesson, D. P., Jones, K. A. & Costa, A.-M. Substituted 1,2-dihydrophthalazines: potent, selective, and noncompetitive inhibitors of the AMPA receptor. J. Med. Chem. 39, 343±346 (1996). 12. Paternain, A. V., Morales, M. & Lerma, J. Selective antagonism of AMPA receptors unmasks kainate recceptor-mediated responses in hippocampal neurons. Neuron 14, 185±189 (1995). 13. Wilding, T. J. & Huettner, J. E. Differential antagonism of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid-preferring and kainate-preferring receptors by 2,3-benzodiazepines. Mol. Pharmacol. 47, 582±587 (1995).

14. Jones, K. A., Wilding, T. J., Huettner, J. E. & Costa, A.-M. Desensitization of kainate receptors by kainate, glutamate and diasteriomers of 4-methylglutamate. Neuropharmacol. 36, 853±863 (1997). 15. Wilding, T. J. & Huettner, J. E. Activation and desensitization of hipocampal kainate receptors. J. Neurosci. 17, 2713±2721 (1997). 16. Partin, K. M., Patneau, D. K., Winters, C. A., Mayer, M. L. & Buonanno, A. Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A. Neuron 11, 1069±1082 (1993). 17. Todd, A. J., Spike, R. C., Price, R. F. & Neilson, M. Immunocytochemical evidence that neurotension is present in glutamatergic neurons in the super?cial dorsal horn of the rat. J. Neurosci. 14, 774±784 (1994). ? ? 18. Tolle, T. R., Berthele, A., Zieglgansberger, W., Seeburg, P. H. & Wisden, W. The differential expression of 16 NMDA and non-NMDA receptor subunits in the rat spinal cord and in periagqueductal gray. J. Neurosci. 13, 5009±5028 (1993). 19. Wisden, W. & Seeburg, P. H. A complex mosaic of high-af?nity kainate receptors in rat brain. J. Neurosci. 13, 3582±3598 (1993). 20. Petralia, R. S., Wang, Y.-X. & Wenthold, R. J. Histological and ultrastructural localization of the kainate receptor subunits, KA2 and GluR6/7, in the rat nervous system using selective antipeptide antibodies. J. Comp. Neurol. 349, 85±110 (1994). 21. Fitzgerald, M., Butcher, T. & Shortland, P. Development changes in the laminar termination of A ?bre cutaneous sensory afferents in the rat spinal cord dorsal horn. J. Comp. Neurol. 348, 225±233 (1994). 22. Coggeshall, R. E., Jennings, E. A. & Fitzgerald, M. Evidence that large myelinated primary afferent ?bers make synaptic contacts in lamina II of neonatal rats. Brain Res. Dev. Brian Res. 92, 81±90 (1996). 23. Baba, H., Yoshimura, M., Nishi, S. & Shimoji, K. Synaptic responses of substantia gelatinosa neurons to dorsal column stimulation in rat spinal cord in vitro. J. Physiol. (Lond.) 478, 87±99 (1994). 24. Huang, L.-Y. M., Carlton, S. M. & Willis, W. D. Identi?cation of spinothalamic tract cells in fresh, un?xed rat spinal cord. J. Neurosci. Methods 14, 91±96 (1985). 25. Zhuo, M. NMDA receptor-dependent long term hyperalgesia after tail amputation in mice. Eur. J. Pharmacol. 349, 211±220 (1998). 26. Cao, Y. Q. et al. Primary afferent tachykinins are required to experience moderate to intense pain. Nature 392, 390±394 (1998). 27. De Felipe, C. et al. Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature 392, 394±397 (1998). 28. Li, P. & Zhuo, M. Silent glutamatergic synapses and nociception in mammalian spinal cord. Nature 393, 695±698 (1998). Acknowledgements. We thank D. Leander for GYKI 53655. This work was supported in part by grants from NIDA (to M.Z.) and NINDS (to J.E.H.) of the NIH. Correspondence and requests for materials should be addressed to M.Z. (e-mail: zhuom@morpheus. wustl.edu).

The oncogene and Polycombgroup gene bmi-1 regulates cell proliferation and senescence through the ink4a locus
Jacqueline J. L. Jacobs*, Karin Kieboom*, Silvia Marino?, Ronald A DePinho? & Maarten van Lohuizen*
* Division of Molecular Carcinogenesis and ? Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands ? Dana Farber Cancer Institute, Harvard Medical School, 44 Binney Street (M463), Boston, Massachusetts 02115, USA
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The bmi-1 gene was ?rst isolated as an oncogene that cooperates with c-myc in the generation of mouse lymphomas1,2. We subsequently identi?ed Bmi-1 as a transcriptional repressor belonging to the mouse Polycomb group3±6. The Polycomb group comprises an important, conserved set of proteins that are required to maintain stable repression of speci?c target genes, such as homeobox-cluster genes, during development7±9. In mice, the absence of bmi-1 expression results in neurological defects and severe proliferative defects in lymphoid cells, whereas bmi-1 overexpression induces lymphomas4,10. Here we show that bmi-1-de?cient primary mouse embryonic ?broblasts are impaired in progression into the S phase of the cell cycle and undergo premature senescence. In these ?broblasts and in bmi-1-de?cient lymphocytes, the expression of the tumour suppressors p16 and p19Arf, which are encoded by ink4a, is raised markedly. Conversely, overexpression of bmi-1 allows ?broblast immortalization, downregulates expression of p16 and p19Arf and, in combination with H-ras, leads to neoplastic transformation. Removal of ink4a dramatically reduces the lymphoid and neurological defects seen in bmi-1NATURE | VOL 397 | 14 JANUARY 1999 | www.nature.com

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de?cient mice, indicating that ink4a is a critical in vivo target for Bmi-1. Our results connect transcriptional repression by Polycomb-group proteins with cell-cycle control and senescence. To investigate the role of bmi-1 in cell proliferation in biochemical detail, we used primary mouse embryonic ?broblasts (MEFs) derived from bmi-1-/- embryos. Bmi-1-/- MEFs have a signi?cantly reduced proliferation rate compared with bmi-1+/+ MEFs (Fig. 1a). This is because of an S-phase defect, as shown by a reduced rate of incorporation of the DNA-labelling dye bromodeoxyuridine (BrdU) (Fig. 1b). Cytoplasmic enlargement, ?attening of the cells, unresponsiveness to growth factors and expression of acidic bgalactosidase11 in 80% of arrested passage 3 bmi-1-/- MEFs indicates premature entry into senescence, in contrast to wild-type cells which ?rst arrest at passage 7 (Fig. 1e). Re-expression of wildtype bmi-1 by retroviral transduction, but not expression of Bmi-1 with a mutation in the RING-?nger domain that is incapable of inducing tumours in vivo10, prevents premature senescence and completely restores the proliferative capacity of bmi-1-/- cells, indicating speci?city of bmi-1-/- arrest (Fig. 1c, d). Wild-type MEFs that overexpress bmi-1 proliferate signi?cantly faster and to higher cell densities than control cells, and have an extended lifespan: they either become immortal immediately or enter a slow growth period at about passage 13, after which they easily become immortalized (Fig. 1c and Supplementary Information). Similar data were obtained using a de?ned 3T3 culture scheme (see Supplementary Information). Thus, lack of bmi-1 expression causes premature entry into senescence, whereas overexpression of bmi-1 allows immortalization. We observed a severe downregulation of cyclin A and cyclin E
BrdU-positive cells (%) b 12 10 8 6 4 2 0 0 2 4 6 8 10 Days in culture

protein levels in bmi-1-/- MEFs, which did not occur in bmi-1-/MEFs infected with the bmi-1 retrovirus (Fig. 2a). Although no differences were observed in amounts of the cyclin-dependent kinase inhibitors p21 and p27 or the tumour suppressor p53, an upregulation of p16 protein in bmi-1-/- MEFs compared with wildtype MEFs was clearly seen (Fig. 2a, b, and data not shown). Conversely, overexpression of bmi-1, but not of the RING-?nger mutant protein, led to rapid downregulation of p16 levels in bmi-1-/MEFs and in wild-type MEFs (Fig. 2a, b). Signi?cantly, p16 is thought to be involved in the induction of replicative senescence12±14. In agreement with the highly conserved structure and function of Polycomb-group (PcG) genes7,8, overexpression of bmi1 also downregulates p16 protein levels in TiG-3 human primary ?broblasts (Fig. 2c). This downregulation is accompanied by delayed entry into senescence, although the bmi-1-overexpressing human cells are not fully immortalized and arrest after 67 more population doublings than normal (Fig. 2d). Human cells show more complex and multifactorial regulation of the senescence checkpoint than murine cells12,13,15,16. In agreement with this we observed no detectable telomerase reactivation upon bmi-1 overexpression in TiG-3 cells using the sensitive telomeric-repeatampli?cation protocol (data not shown). The tumour suppressor p19Arf, which, like p16, is also encoded by the ink4a locus but is controlled by a separate promoter, is also important in cell proliferation and senescence17. p16 and, to a lesser extent, p19Arf steady-state transcript levels were upregulated on average eightfold and two- to threefold, respectively, in bmi-1-/MEFs (Fig. 3a) and splenocytes (Fig. 4a), whereas the neighbouring
+ Bmi-1

Relative number of cells

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1+/+ 3+/+ 4–/– 16–/– 1+/+ 3+/+ 4–/– 16–/–

30 20 10 0 +/+ –/–

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C. D1 C. A C. E CDK4 p21 p16 Tub.

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16 12 8 4 0 0 2 4 6 8 Days in culture C B

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d 6 4 2 0 0 2 4 6 Days in culture +/+ C +/+ B –/– C –/– B

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–/–B

4 3 2 1 0 0 2 4 6 Days in culture

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p21 p16 Tub.

T1

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Figure 2 Effects of absence or overexpression of bmi-1 on cell-cycle-regulatory proteins in primary ?broblasts. a±c, Western blots of cell lysates. a, Cell lysates from control- or LZRS±bmi-1-infected passage 3 bmi-1+/+ and bmi-1-/- MEFs. Figure 1 Impaired proliferation of bmi-1
-/-

MEFs is rescued by wild-type but not

Tubulin (Tub.) serves as loading control. C.D1, cyclin D1; C.A, cyclin A; C.E, cyclin E; CDK4, cyclin-dependent kinase4. b, Cell lysates from control (C) or LZRS±bmi-1 (B)-infected bmi-1+/+ and bmi-1-/- MEFs, showing p16 and p21 expression 48 h (T1) and 6 d (T2) after infection. c, Downregulation of p16 protein in primary human TiG-3 cells after infection with LZRS±bmi-1 virus (B), but not after infection with control (C) or BZN-mutant (Z) virus. U, uninfected. d, Growth curves for TiG-3 cells infected at PDL 50 with control (C) or Bmi-1-expressing (B) retrovirus; growth was monitored for control-infected cells starting at PDL 68 (nearly senescent) and for Bmi-1-overexpressing cells at PDL 70 (still proliferating).

mutant bmi-1. a, Growth curves for two independent bmi-1+/+ and bmi-1-/- MEF cultures at passage 2. The numbers refer to independently derived MEF cultures. b, Level of BrdU incorporation by asynchronously growing passage 3 bmi-1+/+ and bmi-1-/- MEFs. c, d, Growth curves for passage 2 bmi-1+/+ and bmi-1-/- MEFs infected at passage 1 with `empty' control LZRS retrovirus (C), LZRS±bmi-1 virus (B) or LZRS±BZN virus (Z): wild type Bmi-1 rescues proliferation, whereas the BZN Bmi-1 RING-?nger mutant is unable to do so. e, b-Galactosidase (pH 6) activity in passage 1 and 3 bmi-1+/+ and bmi-1-/- MEFs. NATURE | VOL 397 | 14 JANUARY 1999 | www.nature.com

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p15ink4b gene was only modestly affected (1.6-fold upregulation; Fig. 3a). This gradual effect on the ink4a locus is in line with transcriptional repression by PcG proteins in Drosophila, which is thought to spread over a limited distance from cis elements into adjacent chromatin18±20. As PcG proteins act in multiprotein complexes5,21,22, we analysed senescence entry by MEFs de?cient in another PcG gene. Signi?cant upregulation of p16 messenger RNA levels (threefold) was also observed in mel18-/- MEFs23; this upregulation was accompanied by premature senescence entry at passage 5 (Fig. 3b). The passage of arrest correlated well with relative levels of p16 induction. This indicates that the effects on the cell cycle and senescence are intrinsic to the interaction of PcG proteins in repressive complexes. Consistent with transcriptional repression by PcG proteins, downregulation of steady-state levels of p16 (fourfold) and p19Arf (2.4-fold) mRNA were easily detected in bmi-1-infected MEFs, but not in cells infected with control virus (Fig. 4b). Unlike wild-type MEFs but as reported for ink4a-/- MEFs, MEFs overexpressing bmi-1 could be transformed by oncogenic ras alone, as detected by a focus-formation assay; serial dilutions of the bmi-1 retrovirus led to a dose-dependent reduction in the number of transformed foci (Fig. 4c). This indicates that bmi-1 and ras cooperate in neoplastic transformation of primary MEFs; in a similar way, ras and the loss of p16 and p19Arf expression cooperate to induce neoplastic transformation17,24. To determine in vivo to what extent the upregulation of p16 and p19Arf is needed to produce the observed proliferative defects in bmi-1-/- mice, mice heterozygous for the bmi-1 gene and for exons 2 and 3 of ink4a were intercrossed to generate double-knockout mice as well as heterozygous and wild-type control littermates. In the ?rst litters (37 offspring out of 5 litters), we observed two mice of an intermediate size, two very small mice and a large group of mice of normal size. The two small mice developed ataxia at about 3±4 weeks after birth, unlike the intermediate-sized mice. Genotyping con?rmed that the two small mice were bmi-1-/- ink4a+/+ whereas the mice of intermediate size were bmi-1-/- ink4a-/-. At 5 weeks of age, the bmi-1-/- ink4a+/+ mice had progressed to severe ataxia, whereas the two double-knockout mice were still indistinguishable in behaviour from wild-type mice. Histopathological analysis of bmi-1-/- ink4a-/- mice showed a dramatic rescue of the cerebellar defects normally observed in bmi-1-/mice4 (Fig. 5a). The size of the cerebellum was comparable to the wildtype cerebellum, the width of the granular cell layer was restored and the cellularity in the granular and molecular layers increased (Fig. 5b). Notwithstanding the rescued cellularity, the reactive astrogliosis observed in bmi-1-/- mice4 was only slightly reduced in the double-knockout mice (see Supplementary Information). This result indicates that there may be only a partial rescue of the neurological defects, resulting in a delayed onset and slower progression of the pathological process. These results, which are in line with the signi?cant upregulation of p16 and p19Arf expression in RNA isolated from bmi-1-/- brains (data not shown) and with the absence of behavioural disorders in double-knockout mice at 5 weeks of age, indicate that ink4a is a critical target for bmi-1 in this pathological setting too. Signi?cant rescue of thymocyte and splenocyte cell numbers to up to 50±70% of wild-type numbers was seen in bmi-1-/- ink4a-/- mice, in contrast with bmi-1-/- ink4a+/+ littermates which retained only 2±4% of wild-type cell numbers (Fig. 5c). Fluorescence-activated cell sorting (FACS) analysis, using standard T- and B-cell differentiation cell-surface markers, of bmi-1-/- ink4a-/- thymocytes and splenocytes showed pro?les indistinguishable from those of wild-type cells; bmi-1-/- ink4a+/+ littermates, however, showed increased populations of CD4- CD8-, CD3 dull CD25+ or sIg- B220+ immature cells (Fig. 5d). Independent crosses con?rmed the rescue of cerebellar and lymphoid defects in another ink4a-/- bmi-1-/- mouse. Full rescue of the proliferative defects and premature senescence entry of bmi-1-/MEFs was also observed in bmi-1-/- ink4a-/- MEFs, which grew
3,125x 3,125x p15 HPRT p19Arf HPRT p16 HPRT – + b Probe: p15 U C B p19Arf U C B p16 U C B a 0x 5x M 0.5kb 0.2kb 0.5kb 0.2kb 0.5kb 0.2kb

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1:3 Bmi-1 1:6 Bmi-1 Ras Ras

Figure 4 p15, p16 and p19Arf mRNA levels in bmi-1+/+ and bmi-1-/- splenocytes and

a Probe: p15 +/+ –/– +/– p19Arf +/+ –/– +/– p16 +/+
–/– +/–

b Probe: p16 +/+ –/–

control or bmi-1-virus-infected MEFs, and cooperation between Ras and Bmi-1 in neoplastic transformation. a, Semiquantitative RT-PCR analysis of speci?c p15, p16 and p19Arf transcripts in bmi-1+/+ and bmi-1-/- spleens. Simultaneous ampli?cation of hypoxanthine guanine phosphoribosyl transferase (Hprt) served as an internal control. Water (-) and total MEF cDNA (+) also served as controls. M, markers; kb, kilobases. The numbers above the lanes represent serial dilution of ?rst-strand cDNA template used. b, Northern blot analysis of p15,

GAPDH

GAPDH

p19Arf exon 1b and p16 exon 1a transcripts in uninfected (U), control (C) or LZRS±bmi-1-infected (B) bmi-1-/- MEFs. GAPDH served as a loading control c, Focus-formation assay using bmi-1+/+ MEFs infected with `empty' retrovirus (top left) or with retroviruses encoding the proteins indicated. The plates in the top row, except top left, were infected with retroviruses encoding 12S E1A, Ras or Bmi-1, and were superinfected with an `empty' retrovirus. The plates in the bottom row were infected with retroviruses encoding 12S E1A, undiluted Bmi-1, or Bmi-1 diluted 1:3 or 1:6; they were then superinfected with H-ras V12 retrovirus.

Figure 3 p15, p16 and p19Arf mRNA levels in bmi-1+/+, bmi-1-/- and bmi-1+/- MEFs and in mel-18+/+ and mel-18-/- MEFs. a, Northern blot analysis of p15, p19Arf exon 1b and p16 exon 1a transcripts in passage 3 bmi+/+, bmi-1-/- or bmi-1+/- MEFs. b, p16 mRNA levels in passage 3 mel-18+/+ and mel-18-/- MEFs. GAPDH was included as a loading control. All signals were quanti?ed relative to the loading control using a phosphorimager.

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faster, readily bypassed senescence arrest and were indistinguishable from ink4a-/- MEFs24 in this respect (Fig. 5e). The overall improved health status of the double-knockout animals, the normal lymphocyte FACS pro?les and the increased cellularity of the cerebellar layers indicate a remarkable restoration of lymphoid homeostasis and neurological functions. This, together with the results of our studies of MEFs, provides evidence that a
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b MGc 100 Nucleated cells (%) 80 60 40 20 0 Spleen Thymus e Relative cell number 12 8 4 0 0 2 4 6 8 Days in culture P

bmi-1+/+ ink4a+/– bmi-1+/+ ink4a–/– bmi-1–/– ink4a+/+ bmi-1–/– ink4a+/+ bmi-1–/– ink4a–/– bmi-1–/– ink4a–/–

d Thymocytes

bmi-1+/+ ink4a+/– 3.1 81.9
CD8

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sIg

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58.8 8.8

52.5 4.3

bm i-1 ink 4a
–/– +/+ +/+ +/+ +/+ –/– –/– –/– –/– –/–

16

common, critical cell-cycle-control locus (ink4a) lies at the heart of proliferative defects in very different affected cell types in bmi-1-/mice. As expected for a major downstream target, overexpression of either p16 or p19Arf from a retroviral vector in MEFs already overexpressing bmi-1 still causes cell-cycle arrest (data not shown), indicating that bmi-1 overexpression does not lead to major activations of signalling pathways downstream of ink4a. In good agreement with ink4a-mediated arrest acting before M2 crisis, karyotype analysis of arrested bmi-1-/- MEFs showed no obvious abnormalities and no signi?cant reduction in telomere length was observed by ?uorescence in situ hybridization analysis (data not shown). These results, together with our observations of human primary cells, indicate that the wearing away of telomeres or telomerase activity are not involved in the observed ink4a-mediated arrest in bmi-1-/- cells. Our results place bmi-1 at an early step in tumorigenesis as an immortalizing oncogene that is capable of cooperating with H-ras in transformation of primary cells, and provide a convenient in vitro assay for the effects of PcG dose on cell-cycle regulation. Moreover, our results offer a plausible explanation for the strong cooperation between bmi-1 and c-myc or H-ras. Whereas oncogenes such as H-ras, c-myc and E1A induce p19Arf/p53 (refs 25, 26) and/or p16 (ref. 15) in primary cells to prevent immortalization during ectopic mitogenic signalling, bmi-1 acts primarily by suppressing p16/cyclin D/retinoblastoma protein (Rb) and/or p19Arf/MDM2/p53 tumoursuppressor pathways, thereby allowing progression through the cell cycle. Both of these latter tumour-suppressor pathways, when defective, cooperate ef?ciently with c-myc or activated cyclin E in the induction of mouse lymphomas27,28. The ef?cient in vivo cooperation of these oncogenes, and our observations that proliferative defects in c-myc-/- rat TGR-1 cells29 cannot be rescued by overexpression of bmi-1 and that bmi-1-/- MEFs cannot be rescued by c-myc-expressing retroviruses, indicates that these oncogenes may ful?l separate, cooperative functions in immortalization (data not shown). In conclusion, our in vivo and in vitro results identify the tumour suppressors p16 and p19Arf as critical downstream targets for the PcG gene and oncogene bmi-1, with regard to its effects on cell proliferation and senescence. This establishes for the ?rst time, to our knowledge, a connection between PcG-mediated silencing, cellcycle regulation and the senescence checkpoint, and shows that PcG-mediated transcription repression is not only crucial for regulation of Hox genes, but also controls the expression of critical cell-cycle regulators. PcG-dependent regulation of ink4a may re?ect the fact that two of the most frequently disrupted tumour-suppressor pathways30 in a variety of neoplasias, p16/cyclin D/Rb and p19Arf/MDM2/p53, each include a protein encoded by the same gene, ink4a16. Thus a tight and coordinated transcriptional regulation of this locus is required. The signi?cance of regulation of ink4a by bmi-1 is underscored by our observation that this regulation is conserved in primary MEFs and human ?broblasts, indicating that overexpression of Bmi-1 may contribute to human neoplasias that retain wild-type ink4a. M
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Methods
Figure 5 Rescue of bmi-1-/--associated proliferative defects in bmi-1-/- ink4a-/mice and MEFs. a, b, Haematoxilin-stained cerebellar sagittal sections. a, Cerebellum of 5-week-old bmi-1+/+ ink4a+/-, bmi-1-/- ink4a+/+ and bmi-1-/- ink4a-/littermates, showing up to 90% restoration of cellularity in double knockouts. b, Higher magni?cation of molecular (M) and granular (G) layers, showing increased cellularity and more regular spacing of Purkinje cells (P) in bmi-1-/- ink4a-/- mice compared with bmi-1-/- ink4a+/+ mice. c, Per cent nucleated cells in spleen and thymus of mice generated by intercrossing bmi-1+/- ink4a+/- mice. d. Flowcytometric analysis of thymocytes and splenocytes of `wild-type' bmi-1+/+ ink4a+/, bmi-1-/- ink4a+/+ and bmi-1-/- ink4a-/- mice. Note the almost complete restoration to wild-type levels of B- and T-lymphocyte subpopulations in double knockouts. e, Growth curves: bmi-1-/- ink4a-/- MEFs proliferate like bmi-1+/+ ink4a-/- MEFs. NATURE | VOL 397 | 14 JANUARY 1999 | www.nature.com

Cell culture, growth curves and retroviral infection. Cells were maintained

in DMEM medium supplemented with 10% fetal bovine serum (Gibco). Head and organs of day 14.5 embryos were dissected; fetal tissue was rinsed in PBS, minced, rinsed twice in PBS and kept overnight on ice with 100 ml trypsin/ EDTA (Gibco). The next day another 100 ml trypsin/EDTA was added and fetal tissue was incubated for 30 min at 37 8C and subsequently dissociated in medium. After removal of large tissue clumps, the remaining cells were plated out in a 175 cm2 ?ask. After 48 h, con?uent cultures were frozen down. These cells were considered as being passage 1 MEFs. For continuous culturing, MEF cultures were split 1:3, 1 passage being equivalent to 1.8 population doublings (PDLs). TiG-3 primary human diploid ?broblast cultures were split 1:4, 1 passage being comparable to 2 PDLs. For growth curves, 2:5 3 104 cells were
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plated in triplicate into 12-well plates. At various time points cells were stained with crystal violet (Sigma) and the optical density at 590 nm was determined15. Values were normalized to the optical density at day 0 (20 h after plating). Phoenix producer cells were used to generate retroviral stocks as described15. For infection, subcon?uent passage 1 MEF cultures were incubated at 37 8C with viral supernatant in the presence of 4 mg ml-1 polybrene (Sigma). After 6 h the viral supernatant was diluted 1:3 with complete medium and left on the cells for 42 h. Appropriate dilutions of viral supernatants were used such that 100% of MEFs were infected. BrdU-incorporation assay and western blotting. MEFs were grown on coverslips and incubated for 4 h with 10 mM BrdU (Amersham). Cells were washed in PBS, ?xed for 15 min at -20 8C in 5% acetic acid/95% ethanol and incubated in PBS. Fixed cells were incubated for 20 min in 2 M HCl/0.5% Triton-X100, for 30 min in blocking solution (5% fetal calf serum, 5% normal goat serum in PBS/0.02% Triton-X100), for 1 h with 1:10 diluted anti-BrdU monoclonal antibody (DAKO) in blocking solution, and overnight at 4 8C with 1:50 diluted ?uorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Jackson Immuno Research Labs) and 4,-6-diamidino-2-phenylindole (DAPI) in blocking solution; this was followed by 3 washes for 5 min each in PBS/0.02% Triton-X100. Cells were embedded in Vectastain (Vector Labs) and the percentage of BrdU-labelled cells (FITC:DAPI ratio) was quanti?ed using a ?uorescence microscope. For protein analysis, cells were washed with PBS, scraped and lysed on ice in RIPA buffer (150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.0, 0.2 mM phenylmethylsulphonyl?uoride (PMSF), 0.5 mM dithiothreitol (DTT)). Cleared lysates were assayed for protein concentration. Equal amounts of protein were separated on 12.5% SDS±PAGE and transferred to nitrocellulose. Western blot analysis was according to standard methods using enhanced chemiluminescence (Amersham). A list of antisera used is available on request. Expression analysis. Total RNA was extracted using guanidinium thiocyanate, separated on 1.2% agarose, transferred to nitrocellulose and hybridized according to standard procedures with 32P-labelled probes speci?c for exon 1a of mouse p16, for exon 1b of mouse p19Arf, for mouse p15 or for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For semiquantitative reverse transcription with polymerase chain reaction (RT-PCR), ?rst-strand complementary DNA was generated from 1 mg total RNA using Superscript II RT (Gibco) and oligo-dT primer according to the manufacturer's instructions. Primer sequences are available upon request. PCR reactions were performed on ?ve-times serial dilutions of ?rst-strand cDNA in 50 ml containing 1? Taq PCR buffer, 1.5 mM MgCl2, 200 mM dNTPs, 0.5 mM of each of four primers, 1 ml of ?rst-strand cDNA template and 1.25 units of Taq DNA Polymerase (Gibco), using 35 cycles of denaturation (94 8C, 1 min), annealing (60 8C, 45 s) and extension (72 8C, 2 min). Products were resolved on 2% NuSieve agarose gels. +/+/Generation of bmi-1-/- ink4a-/- mice. bmi-1 FVB mice4 and ink4a mice 24 on a mixed 129/Sv; C57BL/6 genetic background were crossed to generate bmi-1+/- ink4a+/- mice, which were subsequently intercrossed to generate double-knockout offspring together with control littermates. Mice were genotyped routinely by PCR or Southern blot analysis of tail DNA. Cell count and ?ow-cytometric analysis. Cell suspensions of lymphoid organs were prepared by mincing the tissue though an open ?lter chamber. Cell suspensions were depleted of erythrocytes and the number of nucleated cells ? was determined with a Casy-1 TT automated cell counter (Schafe, Reutlingen, Germany). Flow cytometry with standard B- and T-cell differentiation was done nearly as described10. Histological analysis. Brains were ?xed in 4% formaldehyde in PBS, paraf?nembedded and cut into 4-mm serial sagittal sections. Sections at different levels were stained with haematoxylin and eosin.
Received 10 September; accepted 12 November 1998. 1. van Lohuizen, M. et al. Identi?cation of cooperating oncogenes in Em-myc transgenic mice by provirus tagging. Cell 65, 735±752 (1991). 2. Haupt, Y., Alexander, W. S., Barri, G., Klinken, S. P. & Adams, J. M. Novel zinc ?nger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in Em-myc transgenic mice. Cell 65, 753±763 (1991). 3. van Lohuizen, M., Frasch, M., Wientjens, E. & Berns, A. Sequence similarity between the mammalian Bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2. Nature 353, 353±355 (1991). 4. van der Lugt, N. M. T. et al. Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the Bmi-1 proto-oncogene. Genes Dev. 8, 757±769 (1994). 5. Alkema, M. J. et al. MPc2, a new murine homologue of the Drosophila Polycomb protein is a member of the mouse Polycomb transcriptional repressor complex. J. Mol. Biol. 273, 993±1003 (1997). 6. Alkema, M. J., van der Lugt, N. M. T., Bobeldijk, R. C., Berns, A. & van Lohuizen, M. Transformation of axial skeleton due to overexpression of Bmi-1 in transgenic mice. Nature 374, 724±727 (1995). 7. Paro, R. Propagating memory of transcriptional states. Trends Genet. 11, 295±298 (1995). 8. van Lohuizen, M. Functional analysis of mouse Polycomb-group genes. Cell. Mol. Life Sci. 54, 71±79 (1998). 9. Gould, A. Functions of mammalian Polycomb group and trithorax group related genes. Curr. Opin. Genet. Dev. 7, 488±494 (1997). 10. Alkema, M. J., Jacobs, H., van Lohuizen, M. & Berns, A. Perturbation of B and T cell development and predisposition to lymphomagenesis in Em-Bmi1 transgenic mice require the Bmi1 RING ?nger. Oncogene 15, 899±910 (1997). 11. Dimri, G. P. et al. A biomarker that identi?es senescent human cells in culture and aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363±9367 (1995). 12. Hara, E. et al. Regulation of p16 CDKN2 expression and its implications for cell immortalisation and senescence. Mol. Cell. Biol. 16, 859±867 (1996). 13. Alcorta, D. A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human ?broblasts. Proc. Natl Acad. Sci. USA 93, 13742±13747 (1996). 14. Zindy, F., Quelle, D. E., Roussel, M. F. & Sherr, C. J. Expression of the p16ink4a tumour suppressor versus other ink4 family members during development and aging. Oncogene 15, 203±211 (1997). 15. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16ink4a. Cell 88, 593±602 (1997). 16. Haber, D. A. Splicing into senescence: the curious case of p16 and p19Arf. Cell 91, 555±558 (1997). 17. Kamijo, T. et al. Tumour suppression at the mouse ink4a locus mediated by the alternative reading frame product p19Arf. Cell 91, 649±659 (1997). 18. Strutt, H., Cavalli, G. & Paro, R. Colocalisation of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression. EMBO J. 12, 3621±3632 (1997). 19. Cavalli, G. & Paro, R. The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93, 505±518 (1998). 20. Pirotta, V. PcG complexes and chromatin silencing. Curr. Opin. Genet. Dev. 7, 249±258 (1997). 21. Alkema, M. J. et al. Identi?cation of Bmi1-interacting proteins as constituents of a multimeric mammalian Polycomb complex. Genes Dev. 11, 226±240 (1997). 22. Gunster, M. J. et al. Identi?cation and characterisation of interactions between the vertebrate Polycomb-group protein BMI1 and the human homologues of Polyhomeotic. Mol. Cell. Biol. 17, 2326±2335 (1997). 23. Akasaka, T. et al. A role for mel-18, a Polycomb group-related vertebrate gene, during the anteroposterior speci?cation of the axial skeleton. Development 122, 1513±1522 (1996). 24. Serrano, M. et al. Role of the ink4a locus in tumour suppression and cell mortality. Cell 85, 27±37 (1996). 25. Zindy, F. et al. Myc signalling via the ARF tumour suppressor regulates p53-dependent apoptosis and immortalisation. Genes Dev. 12, 2424±2433 (1998). 26. de Stanchina, E. et al. E1A signalling to p53 involves the p19ARF tumour suppressor. Genes Dev. 12, 2434±2442 (1998). 27. Blyth, K. et al. Synergy between a human c-myc transgene and p53 null genotype in murine thymic lymphomas: contrasting effects of homozygous and heterozygous p53 loss. Oncogene 10, 1717±1723 (1995). 28. Bodrug, S. E. et al. Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene. EMBO J. 13, 2124±2130 (1994). 29. Mateyak, M. K., Obaya, A. J., Adachi, S. & Sedivy, J. M. Phenotypes of c-Myc-de?cient Rat ?broblasts isolated by targeted homologous recombination. Cell Growth Differ. 8, 1039±1048 (1997). 30. Hall, M. & Peters, G. Genetic alterations of cyclins, cyclin-dependent kinases and cdk inhibitors in human cancer. Adv. Cancer Res. 68, 67±108 (1996). Supplementary information is available on Nature's World-Wide Web site (http://www.nature.com) or as paper copy from the London editorial of?ce of Nature. Acknowledgements. We thank G. Peters for p16 and p15 cDNA plasmids; H. Koseki and J. Deschamps for mel-18-/- embryos; T. Ide for the TiG-3 cells; J. Sedivy for c-myc-/- TGR-1 cells; S. Lowe, B. Amati, C. Sherr, M. Ewen, D. Peeper and R. Bernards for gifts of recombinant retroviral vectors; G. Nolan for providing phoenix eco- and amphotropic packaging cell lines and LZRS-IRES-EGFP retroviral vectors; N. van der Lugt for constructing the LZRS-bmi-1-IRES-EGFP retrovirus; E. de Pauw for telomere FISH analysis; and R. Bernards, A. Berns and W. Voncken for critically reading the manuscript J.J.L.J. and K.K. were supported by a grant of the Dutch Cancer Society (K.W.F.) Correspondence and requests for materials should be addressed to M.v.L. (e-mail: lohuizen@nki.nl).

Robustness in bacterial chemotaxis
U. Alon*?, M. G. Surette?, N. Barkai? & S. Leibler*?
Departments of *Molecular Biology and ?Physics, Princeton University, Princeton, New Jersey 08544, USA ? Department of Microbiology and Infectious Diseases, Calgary, Alberta, Canada T2N 4N1
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Networks of interacting proteins orchestrate the responses of living cells to a variety of external stimuli1, but how sensitive is the functioning of these protein networks to variations in their biochemical parameters? One possibility is that to achieve appropriate function, the reaction rate constants and enzyme concentrations need to be adjusted in a precise manner, and any deviation from these `?ne-tuned' values ruins the network's performance. An alternative possibility is that key properties of
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