p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.     

PKCalpha is involved in phorbol ester TPA-mediated stabilization of p14ARF

We generated A21-13 cells expressing p14(ARF) in the presence of doxycycline in order to examine the stability of p14(ARF) protein. The effects of proteasome inhibitor MG132 on p14(ARF) protein stabilization were detectable using our experimental procedure. Introduction of mutant p53 did not affect MG132-mediated p14(ARF) protein stabilization. We found that phorbol ester TPA (12-o-tetradecanoyl-phorbol 13-acetate) stabilized p14(ARF) protein and that p53 status had no effect on TPA-mediated stabilization. TPA-mediated stabilization was abolished by staurosporine but not by lovastatin or U0126. We further investigated which isoforms of PKC were involved in TPA-mediated p14(ARF) stabilization using short-interference RNA. Knockdown of PKCalpha, but not PKCdelta, attenuated TPA-mediated p14(ARF) stabilization. These findings suggest that PKCalpha is involved in TPA-mediated stabilization of p14(ARF) protein, and this effect of TPA was not affected by the Ras/MAPK pathway or p53 status. Our results are indicative of a novel role of PKC in p14(ARF) protein stability.     

p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei. p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested. Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell,recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression.Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results revealed that, compared with control cells, the WI-38 cells in which p19ARFgene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10-12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells.These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

miR-34a is essential for p19Arf-driven cell cycle arrest

The Arf tumor suppressor gene product, p19^Arf, regulates cell proliferation in incipient cancer cells and during embryo development. Beyond its commonly accepted p53-dependent actions, p19^Arf also acts independently of p53 in both contexts. One such p53-independent effect with in vivo relevance includes its repression of Pdgfrβ, a process that is essential for vision in the mouse. We have utilized cell culture-based and mouse models to define a new role for miR-34a in this process. Ectopic expression of Arf in cultured cells enhanced the expression of several microRNAs predicted to target Pdgfrß synthesis, including the miR-34 family. Because miR-34a has been implicated as a p53-dependent effector, we investigated whether it also contributed to p53-independent effects of p19^Arf. Indeed, in mouse embryo fibroblasts (MEFs) lacking p53, Arf-driven repression of Pdgfrβ and its blockade of Pdgf-B stimulated DNA synthesis were both completely interrupted by anti-microRNA against miR-34a. Ectopic miR-34a directly targeted Pdgfrβ and a plasmid reporter containing wild-type Pdgfrβ 3′UTR sequence, but not one in which the miR-34a target sequence was mutated. Although miR-34a expression has been linked to p53—a well-known effector of p19^Arf—Arf expression and its knockdown correlated with miR-34a level in MEFs lacking p53. Finally, analysis of the mouse embryonic eye demonstrated that Arf controlled expression of miR-34a, and the related miR-34b and c, in vivo during normal mouse development. Our findings indicate that miR-34a provides an essential link between p19^Arf and its p53-independent capacity to block cell proliferation driven by Pdgfrβ. This has ramifications for developmental and tumor suppressor roles of Arf.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.     

Role of the p70 pathway in regulating the actin cytoskeleton and cell migration

We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.     

Association of Human SCFSubunit p19with Interphase Centrosomes and Mitotic Spindle Poles

InSaccharomyces cerevisiae,the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19^SKP1, except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF–E3 ligase complex. Here we show by immunofluorescence microscopy that p19^SKP1localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19^SKP1is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Regulation of CD8 T cell responses to infection with parasitic protozoa

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8⁺ T cell response. This review will highlight some of the key studies that established that CD8⁺ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8⁺ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8⁺ T cell responses are initiated and new insights into their phenotypic plasticity     

A cell-autonomous requirement for Cip/Kip cyclin-kinase inhibitors in regulating neuronal cell cycle exit but not differentiation in the developing spinal cord

Control over cell cycle exit is fundamental to the normal generation of the wide array of distinct cell types that comprise the mature vertebrate CNS. Here, we demonstrate a critical role for Cip/Kip class cyclin-kinase inhibitory (CKI) proteins in regulating this process during neurogenesis in the embryonic spinal cord. Using immunohistochemistry, we show that all three identified Cip/Kip CKI proteins are expressed in both distinct and overlapping populations of nascent and post-mitotic neurons during early neurogenesis, with p27(Kip1) having the broadest expression, and both p57(Kip2) and p21(Cip1) showing transient expression in restricted populations. Loss- and gain-of-function approaches were used to establish the unique and redundant functions of these proteins in spinal cord neurogenesis. Using genetic lineage tracing, we provide evidence that, in the absence of p57, nascent neurons re-enter the cell cycle inappropriately but later exit to begin differentiation. Analysis of p57(Kip2);p27(Kip1) double mutants, where p21 expression is confined to only a small population of interneurons, demonstrates that Cip/Kip CKI-independent factors initiate progenitor cell cycle exit for the majority of interneurons generated in the developing spinal cord. Our studies indicate that p57 plays a critical cell-autonomous role in timing cell cycle exit at G1/S by opposing the activity of Cyclin D1, which promotes cell cycle progression. These studies support a multi-step model for neuronal progenitor cell cycle withdrawal that involves p57(Kip2) in a central role opposing latent Cyclin D1 and other residual cell cycle promoting activities in progenitors targeted for differentiation.     

4-1BB cross-linking enhances the survival and cell cycle progression of CD4 T lymphocytes

4-1BB, a T cell co-stimulatory receptor, prolongs the survival and multiplication of CD4 T cells. Cross-linking 4-1BB stimulated expression of the anti-apoptotic genes bcl-XL and bcl-2, as well as of cyclins D2 and E, and inhibited expression of the cyclin-dependent kinase (cdk) inhibitor p27kip1. Ova-activated CD4 T cells of 4-1BB-deficient/DO11.10 TCR transgenic mice survived less well and underwent less expansion than cells of wild type DO11.10 TCR transgenic mice. These findings demonstrate that 4-1BB is a co-stimulatory molecule for CD4 T cell survival and expansion in vivo.