Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

p53-Dependent and -independent functions of the Arf tumor suppressor.

The Ink4a-Arf locus encodes two closely wedded tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that inhibit cell proliferation by activating Rb and p53, respectively. With few exceptions, the Arf gene is repressed during mouse embryonic development, thereby helping to limit p53 expression during organogenesis. However, in adult mice, sustained hyperproliferative signals conveyed by somatically activated oncogenes can induce Arf gene expression and trigger a p53 response that eliminates incipient cancer cells. Disruption of this tumor surveillance pathway predisposes to cancer, and inactivation of INK4a- ARF by deletion, silencing, or mutation has been frequently observed in many forms of human cancer. Although it is accepted that much of Arf's tumor-suppressive activity is mediated by p53, more recent genetic evidence has pointed to additional p53- independent functions of Arf, including its ability to inhibit gene expression by a number of other transcription factors. Surprisingly, the enforced expression of Arf in mammalian cells promotes the sumoylation of several Arf-interacting proteins, implying that Arf has an associated catalytic activity. We speculate that transcriptional down-regulation in response to Arf-induced sumoylation may account for Arf's p53-independent functions.

Dmp1 is haplo-insufficient for tumor suppression and modifies the frequencies of Arf and p53 mutations in Myc-induced lymphomas.

Loss of Dmp1, an Arf transcriptional activator, leads to spontaneous tumorigenesis in mice, causing death from various forms of cancer by two years of age. Retention and expression of the wild-type Dmp1 allele in tumors arising in Dmp1(+/-) mice demonstrate that Dmp1 can be haplo-insufficient for tumor suppression. The mean latency of E(mu)-Myc-induced B-cell lymphomas is halved on a Dmp1(-/-) or Dmp1(+/-) genetic background. Although p53 mutations or Arf deletion normally occur in approximately 50% of E(mu)-Myc-induced lymphomas, Dmp1 loss obviates selection for such mutations, indicating that Dmp1 is a potent genetic modifier of the Arf-p53 pathway in vivo.

Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either the Arf tumor suppressor or its downstream target, the p53 transcription factor. Mouse p19(Arf) is encoded by the Ink4a-Arf locus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16(Ink4a). We surveyed bone marrow-derived cells from wild-type, Ink4a-Arf-null, or Arf-null mice for their ability to bypass senescence during continuous passage in culture. Unlike preB cells from wild-type mice, those from mice lacking Arf alone could be propagated indefinitely when placed onto stromal feeder layers engineered to produce IL-7. The preB cell lines remained diploid and IL-7-dependent and continued to express elevated levels of p16(Ink4a). By contrast, Arf-null bone marrow-derived macrophages that depend on colony-stimulating factor-1 for proliferation and survival in culture initially grew at a slow rate but gave rise to rapidly and continuously growing, but still growth factor-dependent, variants that ceased to express p16(Ink4a). Wild-type bone marrow-derived macrophages initially expressed both p16(Ink4a) and p19(Arf) but exhibited an extended life span when p16(Ink4a) expression was extinguished. In all cases, gene silencing was accompanied by methylation of the Ink4a promoter. Therefore, whereas Arf loss alone appears to be the major determinant of establishment of murine fibroblast and preB cell lines in culture, p16(Ink4a) provides an effective barrier to immortalization of bone marrow-derived macrophages.

c-Myc-mediated regulation of telomerase activity is disabled in immortalized cells.

Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), as ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation.

We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19INK4D and p21CIP1 in macrophages. IL-10 cannot induce p19INK4D expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10Ralpha which does not recruit Stat3 in J774 cells, whereas p21CIP1 induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19INK4D expression. Sequencing of the murine p19INK4D promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19INK4D mediates inhibition of proliferation. Enforced expression of murine p19INK4D cDNA J774 cells significantly reduced their proliferation. Use of antisense p19INK4D and analysis of p19INK4D-deficient macrophages confirmed that p19INK4D is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19INK4D and Stat3-independent induction of p21CIP1 are important components of the mechanism by which IL-10 blocks proliferation in macrophages.

INK4d-deficient mice are fertile despite testicular atrophy.

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases.

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19(Ink4d) and p27(Kip1), in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G(2) and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19(Ink4d) and p27(Kip1) cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Loss of the ARF tumor suppressor reverses premature replicative arrest but not radiation hypersensitivity arising from disabled atm function.

The alternative reading frame product (p19ARF) of the mouse INK4a/ARF locus is induced by oncoproteins such as Myc and E1A as part of a checkpoint response that limits cell cycle progression in response to hyperproliferative signals. ARF binds directly to Mdm2 to prevent down-regulation of p53 and thereby promotes p53-dependent transcription and cell cycle arrest. However, ARF is not required for p53 induction in response to ionizing radiation or other forms of DNA damage. Animals lacking a functional ataxia telangiectasia (Atm) gene are exquisitely sensitive to ionizing radiation; Atm-null mouse embryo fibroblasts (MEFs) undergo premature replicative arrest, which is relieved by the loss of p53. Here we show that the loss of ARF expands the life expectancy of Atm-null MEFs, but alters neither the sensitivity of Atm-null mice to ionizing radiation nor their propensity to develop lymphomas early in life. Therefore, whereas ARF and Atm signal to p53 through distinct pathways, the loss of ARF can modify p53-dependent features of the Atm-null phenotype.

Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization.

Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.

E1A signaling to p53 involves the p19(ARF) tumor suppressor.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.

Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.

The INK4a-ARF locus encodes two proteins, p16(INK4a) and p19(ARF), that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19(ARF) can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21(Cip1), and to arrest cell proliferation. Binding of p19(ARF) to p53 requires the ARF N-terminal domain (amino acids 1-62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19(ARF) in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to approximately 75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19(ARF) restores p53's ability to induce p21(Cip1) and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.

Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.

The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF+/+ or ARF+/- MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.

Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging.

Four INK4 proteins can prevent cell proliferation by specifically inhibiting cyclin D-dependent kinases. Both p18INK4c and p19INK4d were widely expressed during mouse embryogenesis, but p16INK4a and p15INK4b were not readily detected prenatally. Although p15INK4b, p18INK4c and p19INK4d were demonstrated in many tissues by 4 weeks after birth, p16INK4a protein expression was restricted to the lung and spleen of older mice, with increased, more widespread mRNA expression during aging. Transcripts encoding the INK4a alternative reading frame product p19ARF were not detected before birth but were ubiquitous postnatally. Expression of p16INK4a and p15INK4b was induced when mouse embryos were disrupted and cultured as mouse embryo 'fibroblasts' (MEFs). The levels of p16INK4a and p18INK4c, but not p15INK4b or p19INK4d, further increased as MEFs approached senescence. Following crisis and establishment, three of four independently-derived cell lines became polyploid and expressed higher levels of functional p16INK4a. In contrast, one MEF line that sustained bi-allelic deletions of INK4a initially remained diploid. Therefore, loss of p16INK4a and other events predisposing to polyploidy may represent alternative processes contributing to cell immortalization. Whereas p18INK4c and p19INK4d may regulate pre- and postnatal development, p16INK4a more likely plays a checkpoint function during cell senescence that underscores its selective role as a tumor suppressor.

Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway.

The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination in an in vitro system or in vivo, and it is markedly stabilized (t1/2 approximately 3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells, and although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.

Expression of INK4 inhibitors of cyclin D-dependent kinases during mouse brain development.

In situ hybridization of mouse embryo sections demonstrated expression of mRNAs encoding two polypeptide inhibitors (p18INK4c and p19INK4d) of cyclin D-dependent kinase (CDK) 4 and CDK6 in the central nervous system. No expression of two other INK4 members, p16INK4a and p15INK4b, was observed. The p19INK4d and p18INK4c proteins formed complexes with either CDK4 or CDK6 in a temporal pattern consistent with the results of in situ hybridization. Expression of INK4c was observed at embryonic day 13.5 in neuroepithelial zones of the developing brain, being restricted to dividing neuroblasts but absent from differentiating postmitotic neurons. In the neocortex, p18INK4c was expressed precisely at those developmental stages when neuroblasts switch from a symmetric to an asymmetric pattern of cell division with concomitant increases in their G1 interval. INK4d RNA was detected from embryonic day 11.5 onward, at higher levels than INK4c and with a distinctly different spatial and temporal pattern. Marked INK4d expression was seen in dorsal root ganglia, spinal cord, and focally throughout the brain, but primarily in postmitotic neurons. Neural expression of INK4d continued postnatally into adulthood in postmitotic cells of the dentate gyrus, the pyramidal layer of the hippocampus, and in discrete regions of the cerebral cortex, cerebellum, thalamus, and brainstem. Downregulation of p19INK4d in the dentate gyrus after kainic acid-induced seizures indicated that its expression could also be modified in nondividing cells by excitotoxic stress. Therefore, p19INK4d may contribute to maintaining the quiescent state, acting as a buffer to prevent reactivation of cyclin D-dependent kinases in terminally differentiated cells.

Identification of cyclin A as a molecular target of antinuclear antibodies (ANA) in hepatic and non-hepatic autoimmune diseases.

BACKGROUND/AIMS: Antinuclear antibodies (ANA) are a diagnostic hallmark of various autoimmune diseases and also of autoimmune hepatitis type 1. The designation ANA describes a heterogeneous group of autoantibodies. In liver diseases, only a few nuclear target antigens have been molecularly identified and characterized. Cyclins play a central role in cell cycle regulation, DNA transcription, and cell proliferation. Cyclin A was also identified as an integration site of the hepatitis B virus in a patient with hepatocellular carcinoma. In this study we identify cyclin A as a novel nuclear target protein of ANA. METHODS: Sera of patients with autoimmune hepatitis (AIH) type 1 (n = 61), type 2 (n = 21), and type 3 (n = 39), primary biliary cirrhosis (PBC) (n = 107), rheumatic diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD)) (n = 42) and normal controls (n = 100) were evaluated for ANA by indirect immunofluorescence. Baculovirus-generated recombinant human cyclin A protein was used for immunoblotting to study the prevalence of anti-cyclin A autoantibodies in these sera. RESULTS: Sera of patients with AIH type 1 and rheumatic diseases had ANA detected by indirect immunofluorescence. In AIH type 1 12/61 (20%) and in rheumatic diseases 6/42 (14%) were immunoblot positive for autoantibodies against human cyclin A. In PBC, AIH type 3 and normal control sera negative for ANA by immunofluorescence, anti-cyclin A autoantibodies were present in 7-9%; in AIH type 2 and SLE they were undetectable by immunoblot. In some sera a typical cyclin A immunofluorescence was observed. Anti-cyclin A antibodies recognize a 45 and 50 kDa recombinant protein species, providing evidence for the recognition of at least two molecular epitopes. CONCLUSIONS: This study has identified cyclin A as a human autoantigen in hepatic and non-hepatic autoimmune diseases. More studies are required to evaluate the clinical and pathophysiological significance of anti-cyclin A autoantibodies. The identification of human anti-cyclin A autoantibodies may additionally become a valuable tool for studying the function and regulation of cyclin A in mammalian and human cells.

Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.

The INK4a (MTS1, CDKN2) gene encodes an inhibitor (p16INK4a) of the cyclin D-dependent kinases CDK4 and CDK6 that blocks them from phosphorylating the retinoblastoma protein (pRB) and prevents exit from the G1 phase of the cell cycle. Deletions and mutations involving INK4a occur frequently in cancers, implying that p16INK4a, like pRB, suppresses tumor formation. An unrelated protein (p19ARF) arises in major part from an alternative reading frame of the mouse INK4a gene, and its ectopic expression in the nucleus of rodent fibroblasts induces G1 and G2 phase arrest. Economical reutilization of coding sequences in this manner is practically without precedent in mammalian genomes, and the unitary inheritance of p16INK4a and p19ARF may underlie their dual requirement in cell cycle control.

Overexpression of human cyclin A advances entry into S phase.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian cells. Using a genomic construction corresponding to the human cyclin A gene under the control of its own promoter, we have established stable transfectants overexpressing cyclin A protein. Experiments assisted by laser scanning image cytometry showed that this overexpression begins from late G1 phase onwards and is therefore cell cycle-regulated in this model. We demonstrated that this overexpression advances entry into S phase, leading to a contraction of the overall cell generation time. These results provide evidence that cyclin A can be a rate-limiting factor with respect to the control of the transition to S phase in mammalian cells.