The oncogenic potential of Kaposi's sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo

Expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) cyclin D homolog, K cyclin, is thought to contribute to viral oncogenesis. We show that K cyclin expression in primary cells sensitizes to apoptosis and induces growth arrest, both of which are dependent on p53 but independent of E2F1 or p19(ARF). DNA synthesis, but not cytokinesis, continues in K cyclin-expressing cells, leading to multinucleation and polyploidy. Such polyploid cells exhibit pronounced centrosome amplification and consequent aneuploidy. Our data suggest that K cyclin expression leads to cytokinesis defects and polyploidy, which activates p53. However, in the absence of p53, such cells survive and expand as an aneuploid population. Corroborating these findings, in vivo Emu; K cyclin expression cooperates with p53 loss in the induction of lymphomas.     

Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations

Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.     

The role of Ink4a/Arf in ErbB2 mammary gland tumorigenesis

Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs in < or =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.     

Elevated Mdm2 expression induces chromosomal instability and confers a survival and growth advantage to B cells

Mdm2, a regulator of the p53 tumor suppressor, is frequently overexpressed in lymphomas, including lymphomas that have inactivated p53. However, the biological consequences of Mdm2 overexpression in lymphocytes are not fully resolved. Here, we report that increased expression of Mdm2 in B cells augmented proliferation and reduced susceptibility to p53-dependent apoptosis, which was due to inhibition of p53 and suppression of p21 expression. Notably, developing and mature B cells from Mdm2 transgenic mice had an increased frequency of chromosomal/chromatid breaks and/or aneuploidy. This Mdm2-mediated genome instability occurred at a similar frequency as that in B cells overexpressing the oncogene c-Myc, but the chromosomal instability was not further enhanced when Mdm2 and c-Myc were overexpressed together. Elevated Mdm2 expression alone increased the occurrence of B-cell transformation in vivo and cooperated with c-Myc overexpression, resulting in an acceleration of B-cell lymphomagenesis. In addition, the frequency of p53 mutations was reduced, but not eliminated, in lymphomas arising in Mdm2/Emu-myc double transgenic mice. Therefore, increased Mdm2 expression facilitated B-cell lymphomagenesis, in part, through regulation of p53 by altering B-cell proliferation and susceptibility to apoptosis, and by inducing chromosomal instability.     

Loss of CBP causes T cell lymphomagenesis in synergy with p27Kip1 insufficiency

CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.     

A mouse model for cyclin E-dependent genetic instability and tumorigenesis

Ubiquitination of murine cyclin E is triggered by phosphorylation on threonine 393. Cyclin E(T393A) knockin mice exhibited increased cyclin E stability, but no phenotypic abnormalities. Importantly, loss of the p53 pathway exacerbated the effect of the T393A mutation. Thus, in p21(-/-) cells the T393A mutation had an exaggerated effect on cyclin E abundance and its associated kinase activity, which caused abnormal cell cycle progression, and genetic instability involving chromosome breaks and translocations. Moreover, cyclin E(T393A) acted synergistically with p53 deficiency to accelerate tumorigenesis in cyclin E(T393A) p53(-/-) mice; Ras more readily transformed cyclin E(T393A) p53(-/-) cells than p53(-/-) cells in vitro; and cyclin E(T393A) mice had a greatly increased susceptibility to Ras-induced lung cancer.     

Protein levels of p21, p27, cyclin E and Bax predict sensitivity to cisplatin and paclitaxel in head and neck squamous cell carcinomas

Regression of tumor mass by chemotherapy is caused by growth suppression and/or apoptosis of tumor cells. Therefore, expression levels of cell cycle molecules and apoptosis should be predictive markers for the efficacy of a drug. In the present study, the relationship between expression of molecules in the cell cycle and apoptosis and chemosensitivity was investigated in head and neck squamous cell carcinoma cell lines. Expression of p53, p21, p27, cyclin D1, cyclin E, and Bax in 17 such cell lines were analyzed by Western blot analysis. The concentrations of four chemotherapeutic agents (cisplatin, 5-FU, vincristine, and paclitaxel) resulting in 50% cell growth inhibition were calculated as IC50 values for each cell line. Cell cycle analysis was performed using a FACScan flow cytometer. Cells with strong expression of p21, p27, or Bax showed significantly higher sensitivity to cisplatin, and cells with strong expression of Bax or weak expression of cyclin E showed significantly higher sensitivity to paclitaxel. Cisplatin most effectively killed cells expressing both p21 and p27 or either at G1 phase. Though the assessments of p21, p27, Bax, and cyclin E expression in tumor tissues have been reported to be useful as prognostic factors in head and neck squamous cell carcinoma, these correlations might not only describe the malignant biological behavior of the tumor, but also the response to chemotherapy. Furthermore, p21/p27 expression might be a useful guide for the choice of chemotherapeutic agents.     

褪黑素对小鼠肝癌H22细胞抗增殖和促凋亡的p53依赖性机制研究

背景与目的：本室已经证明了褪黑素(melatonin,MLT)对小鼠肝癌H22细胞的生长具有抑制作用,本研究旨在探讨其机理。方法：(1)建立荷瘤小鼠模型并给予褪黑素处理,取肿瘤组织进行p53、cyclin E水平的免疫组化分析;(2)不同浓度褪黑素体外培养小鼠肝癌H22细胞,采用流式细胞技术(flow cytometry,FCM)检测细胞周期分布和凋亡率;免疫组化法分析培养细胞的p53和cyclin E的蛋白水平;采用RT-PCR方法检测Fas mRNA的水平。结果：(1)FCM显示褪黑素可引起H22细胞G0／G1期比例升高,从75．24％上升至85．46%,同时细胞凋亡增多,从5．07％升至12．77％;(2)褪黑素作用后p53水平较对照组增高42．5％(培养细胞)和19．5%(肿瘤组织),而cyclin E的水平则降低31．7％(培养细胞)和39．9％(肿瘤组织);(3)RT-PCR结果显示Fas mRNA水平增高44、2％。结论：MLT通过阻滞细胞周期和诱导细胞凋亡抑制H22细胞增殖。其机理可能是通过p53抑制下游的cyclin E的表达,阻止细胞进入S期;同时又通过p53上调Fas表达,从而诱导细胞凋亡。     

Loss of p27(Kip1) cooperates with cyclin E in T-cell lymphomagenesis

Cyclin E and p27(Kip1) are key regulators for cyclin-dependent kinases (Cdks) acting at the G1-/S-phase transition of the cell cycle. Whereas cyclin E is required for the activation of Cdk2, p27(Kip1) is a specific Cdk inhibitor and can block cell division. High levels of cyclin E and low levels of p27(Kip1) expression have been associated with malignant lymphomas in humans; the level of p27(Kip1) is even considered of prognostic significance. However, mice that lack p27(Kip1) do not develop any malignant lymphomas despite a pronounced lymphoid hyperplasia in thymus and spleen. We have previously described transgenic mice that carry a construct in which the cyclin E cDNA is under the control of the CD2 promoter/enhancer region and thus express high levels of cyclin E in the T-cell compartment (CD2-cyclin E). These animals are not predisposed for T-cell lymphomas in the absence of other cooperating events. Here we show that T-cells from CD2-cyclin E mice that at the same time are deficient for p27(Kip1) show a significantly higher Cdk2 activity than cells from wild-type or single mutant animals. Accordingly, a higher percentage of T cells in S/G2/M phase is found in CD2-cyclin E/p27(Kip1-/-) mice. After a long latency period of over 200 days, these animals develop spontaneous monoclonal T cell lymphoma whereas none of the single CD2-cyclin E transgenic or the p27(Kip1)-deficient mice showed any sign of lymphoid malignancies. Our findings demonstrate that a deregulation of control mechanisms at the G1/S transition by the combination of high cyclin E levels in the absence of p27(Kip1) is sufficient to predispose mice to develop lymphoid malignancies and further support a role of p27(Kip1) as a tumor suppressor and of cyclin E as a dominant oncogene.     

Bcl-2 and p53 expressions in large-cell lymphoma

Bcl-2 and p53 regulate apoptosis in a see-saw fashion and are expressed abnormally in a variety of malignant tumors. We investigated the frequency and prognostic significance of both bcl-2 and p53 expression in 31 large-cell lymphomas. Immunoreaction of bcl-2 and p53 was found in 74% (23/31) and 65% (20/31) of large-cell lymphomas, respectively. Overall survival was not significantly associated with immunoreactivity for either bcl-2 or p53. Frequent association with both p53 and bcl-2 in large-cell lymphomas confirms the existence of a multistep genetic deregulation in lymphomagenesis, but their clinical significance remains to be elucidated in a larger study.     

Suppression of thymic lymphomas and increased nonthymic lymphomagenesis in Trp53-deficient mice lacking inducible nitric oxide synthase gene

Trp53-deficient mice spontaneously develop lymphomas, mainly of thymic origin, although the molecular mechanism remains largely unknown. As several interaction effects between p53 and iNOS have been reported, we hypothesized that iNOS activity in the thymus is causally linked to lymphomagenesis in Trp53-deficient mice. We therefore created mouse strains with different combinations of the Trp53 and iNOS genes. Western blot and histologic analyses showed that the iNOS protein was constitutively expressed in the thymus independently of Trp53 status and its expression was enhanced in Trp53+/- and Trp53-/- mice compared to Trp53+/+ mice. Homozygous disruption of iNOS decreased the incidence of thymic lymphomas by almost 40% (p=0.087) and 90% (p<0.05) in Trp53-/- and Trp53+/- mice, respectively, compared to the respective iNOS wild-type mice but significantly (p<0.05) increased the development of nonthymic lymphomas in Trp53-/- and Trp53+/- mice. Although iNOS gene disruption did not affect the phenotype of thymic lymphomas, absence of the iNOS gene shifted the spectrum of nonthymic lymphoma from the B-cell to the T-cell lineage. RT-PCR analysis revealed enhanced expression of IL-10, which could have a promoting effect on lymphomagenesis, even without any stimulation, in the spleen of aging mice with the gene combinations Trp53-/-iNOS-/- and Trp53+/-iNOS-/- but not Trp53-/-iNOS+/+ or Trp53+/-iNOS+/+. These results suggest that iNOS could increase the development of thymic lymphomas in Trp53-deficient mice. While iNOS may have protective effects against nonthymic lymphomagenesis, the regulation of cytokine production by iNOS may be involved in the underlying mechanism of antilymphomagenesis effects in the peripheral lymphoid organ.     

苦参碱对白血病细胞癌基因和细胞周期调控蛋白表达的影响

目的：探讨在苦参碱诱导下,白血病细胞株K562增殖和分化时相关癌基因及细胞周期调控蛋白表达表达的改变及意义。方法：应用分子生物学Norhern Blot和DotBlot杂交技术检测人红白细胞病细胞株K562在苦参碱作用后的c-myc,N-ras及p53mRN表达;同时用Western BLotting-ECL分析苦参碱作用前、后24、48、72小时K562细胞Cyclin D1,CyclinE,Ddk2,Cdk5的不同表达水平。结果：在增殖的K562细胞（培养48小时）中,伴随着DNA合成增加,CyclinE和Cdk2保持高表达;而在苦参碱作用24小时分化启动的细胞中,随着DNA合成减少,N-ras,p53mRNA表达增强;c-myc mRNA表达明显受抑;同时CyclinD,Cdk5表达增强,结论：若参碱抑制K562细胞增殖并诱导分化可能与相关癌基因表达和CyclinD1/Cdk5,CyclinE/Cdk2不同表达有关。     

Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo

The phosphatidylinositol-3-OH kinase [PI(3)K] pathway is frequently activated in human cancers and represents a rational target for therapeutic intervention. We have previously shown that enforced expression of Akt, which is a downstream effector of PI(3)K, could promote tumorigenesis and drug resistance in the Emu-myc mouse lymphoma model, and that these tumors were particularly sensitive to inhibition of mammalian target of rapamycin (mTOR) with rapamycin when combined with conventional chemotherapy. We now show that reduced dosage of PTEN, a negative regulator of PI(3)K signaling, is sufficient to activate Akt, but has only a modest effect on lymphomagenesis in the same model. Nonetheless, loss of even one PTEN allele resulted in lymphomas that were resistant to conventional chemotherapy yet sensitive to rapamycin/chemotherapy combinations. These effects could be recapitulated by using RNA interference to suppress PTEN expression in lymphomas, which were previously established in the absence of PI(3)K lesions. Finally, the introduction of lesions that act downstream of mTOR (eIF4E) or disable apoptosis (Bcl-2 and loss of p53) into PTEN+/- lymphomas promoted resistance to rapamycin/chemotherapy combinations. Thus, whether activation of the PI(3)K pathway confers sensitivity or resistance to therapy depends on the therapy used as well as secondary genetic events. Understanding these genotype-response relationships in human tumors will be important for the effective use of rapamycin or other compounds targeting the PI(3)K pathway in the clinic.     

Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228

BACKGROUND: Histone deacetylases (HDACs) modulate chromatin structure by regulating acetylation of core histone proteins. HDAC inhibitors, such as depsipeptide FR901228 (FK228), induce growth arrest and apoptosis in a variety of human cancer cells by mechanisms that cannot be attributed solely to histone acetylation. This study evaluated the mechanisms by which FK228 mediates apoptosis in non-small-cell lung cancer (NSCLC) cells. METHODS: Proliferation and apoptosis were assessed in a panel of NSCLC cell lines that vary in the expression of the growth-regulating proteins p53, pRb, and K-Ras treated with a clinically relevant dose of FK228 (25 ng/mL). Western blot and immunoprecipitation techniques were used to analyze expression of cell-cycle proteins (cyclin A, cyclin E, p53, and p21), signaling-related proteins (ErbB1, ErbB2, and Raf-1), activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2), binding of mutant p53 and Raf-1 to heat shock protein (Hsp)90, and acetylation of Hsp90. RESULTS: FK228 treatment inhibited growth and induced apoptosis in NSCLC cells expressing wild-type or mutant p53. FK228 treatment led to altered expression of cyclin A, cyclin E, and p21, and to reduced expression of mutant, but not wild-type, p53. FK228-treated cells also were depleted of ErbB1, ErbB2, and Raf-1 proteins, and exhibited lower ERK1/2 activity. FK228 treatment also inhibited the binding of mutant p53 and Raf-1 to Hsp90; this inhibition was associated with acetylation of Hsp90. CONCLUSIONS: FK228 depletes the levels of several oncoproteins that are normally stabilized by binding to Hsp90 in cancer cells. The resulting ability of FK228 to diminish signal transduction via pathways involving Raf-1 and ERK may contribute to the potency and specificity of this novel antitumor agent.     

A non-transgenic mouse model for B-cell lymphoma: in vivo infection of p53-null bone marrow progenitors by a Myc retrovirus is sufficient for tumorigenesis

The c-Myc oncoprotein is strongly implicated in B-cell neoplasms such as human Burkitt lymphomas and mouse plasmocytomas. Transgenic mice in which the myc gene is juxtaposed to an immunoglobulin enhancer (E(mu)-myc) also develop B-cell lymphomas, but relatively late in life. In addition, these neoplasms are invariably clonal, suggesting the involvement of additional mutations. Such mutations frequently affect the p53 tumour suppressor gene or its positive regulator Arf, hinting that inactivation of the p53 pathway might be the second hit required for the progression towards malignancy. However, even tumours arising in E(mu)-myc/Arf-null animals are thought to be clonal. This observation raised doubts whether overexpression of Myc in p53-null B-cell precursors is sufficient for tumorigenesis. To address this question, we have established a new, non-transgenic mouse model of B-lymphoma. This model is based on isolation of primary bone marrow (BM) cells, admixing them with packaging cells producing a Myc-encoding retrovirus (LMycSN), and subcutaneous injection into a host with which BM cells are syngeneic. Predictably, wild type BM cells infected in vivo by LMycSN were not tumorigenic. However, LMycSN-infected p53-null BM cells readily gave rise to B-cell lymphomas composed predominantly of late pro-B/small pre-B-cells. In these tumours, heavy chain gene rearrangements were analysed using two independent PCR-based assays. All neoplasms with DJ-rearrangements were found to be polyclonal. This result suggests that inactivation of p53 and overexpression of Myc is all that is necessary for the development of full-fledged B-lymphomas. Our model would also be instrumental in assessing the transforming potential of Myc mutants and in studying cooperation between Myc and other oncogenes.     

BCR signals target p27(Kip1) and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells.

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.