Tumor Suppressor Gene Product Research Articles

Erbin and the NF2 Tumor Suppressor Merlin Cooperatively Regulate Cell-Type-Specific Activation of PAK2 by TGF-β

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

High frequency of loss of allelic integrity at Wilms′ tumor suppressor gene-1 locus in advanced breast tumors associated with aggressiveness of the tumor

The product of Wilms' tumor suppressor gene (WT1), a nuclear transcription factor, regulates the expression of the insulin-like growth factor (IGF) and transforming growth factor (TGF) systems, both of which are implicated in breast tumorigenesis and are known to facilitate angiogenesis. In the present study, WT1 allelic integrity was examined by Loss of Heterozygosity (LOH) studies in infiltrating breast carcinoma (n=60), ductal carcinoma in situ (DCIS) (n=10) and benign breast disease (n=5) patients, to determine its possible association with tumor progression. LOH at the WT1 locus (11p13) as determined by PCR-RFLP for Hinf1 restriction site and was subsequently examined for its association with intratumoral expression of various growth factors i.e. TGF-beta1, IGF-II, IGF-1R and angiogenesis (VEGF and Intratumoral micro-vessel density) in breast carcinoma. Six of 22 (27.2%) genetically heterozygous of infiltrating breast carcinoma and 1 of 4 DCIS cases showed loss of one allele at WT1 locus. Histologically, the tumors with LOH at WT1 were Intraductal carcinoma (IDC) and were of grade II and III. There was no correlation in the appearance of LOH at WT1 locus with age, tumor stage, menopausal status, chemotherapy status and lymph node metastasis. The expression of factor IGF-II and its receptor, IGF-1R was significantly higher in carcinoma having LOH at WT1 locus. A positive correlation was observed between the TGF-beta1, VEGF expression and IMD scores in infiltrating carcinoma. The current study indicates that the high frequency of loss of allelic integrity at Wilms' tumor suppressor gene-1 locus in high-graded breast tumors is associated with aggressiveness of the tumor.

Expression of p53-Regulated Genes in Cultured Mammalian Cells After Exposure to A Space Environment

The space environment contains two major biologically significant influences; space radiation and microgravity. The tumor suppressor gene product p53 is generally thought to contribute to the genetic stability of cells with DNA damage through the activity of p53 initiated signal transduction pathways. We propose to investigate the expression of p53-regulated genes in cultured mammalian cells after exposure to a space environment. We expect that the data from this proposal will be useful for designing physiological protection against the serious effects of space radiation during long-term stays in space.

Resveratrol addiction: To die or not to die

Resveratrol, a polyphenol derived from red grapes, berries, and peanuts, has been shown to mediate death of a wide variety of cells. The mechanisms by which resveratrol mediates cell death include necrosis, apoptosis, autophagy, and others. While most studies suggest that resveratrol kills tumor cells selectively, evidence is emerging that certain normal cells such as endothelial cells, lymphocytes, and chondrocytes are vulnerable to resveratrol. Cell killing by this stilbene may be mediated through any of numerous mechanisms that involve activation of mitochondria and of death caspases; upregulation of cyclin-dependent kinase inhibitors, tumor suppressor gene products, or death-inducing cytokines and cytokine receptors; or downregulation of cell survival proteins (survivin, cFLIP, cIAPs, X-linked inhibitor of apoptosis protein (XIAP), bcl-2, bcl-XL) or inhibition of cell survival kinases (e.g., mitogen-activiated protein kinases (MAPKs), AKT/phosphoinositide 3-kinase (PI3K), PKC, EGFR kinase) and survival transcription factors (nuclear factor-kappaB (NF-kappaB), activating protein 1 (AP-1), HIF-1alpha, signal transducer and activator of transcription (STAT3)). Induction of any of these pathways by resveratrol leads to cell death. While cell death is a hallmark of resveratrol, this polyphenol also has been linked with suppression of inflammation, arthritis, and cardiovascular diseases and delaying of aging. These attributes of resveratrol are discussed in detail in this review.

Prolactin Blocks Nuclear Translocation of VDR by Regulating Its Interaction with BRCA1 in Osteosarcoma Cells

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.

The common mechanisms of transformation by the small DNA tumor viruses: The inactivation of tumor suppressor gene products: p53

The small DNA tumor viruses, Polyoma virus, Simian Vacuolating Virus 40, the Papilloma viruses and the human Adenoviruses, were first described during a period of intense virus discovery (1930–1960s) and shown to produce tumors in animals. In each of these cases the viral DNA was shown to persist (commonly integrated into a host chromosome) and only a selected portion of this DNA was expressed as m-RNA and proteins in these cancers. The viral encoded tumor antigens were identified and shown to be required to both establish the tumor and maintain the transformed cell phenotype. The functions of these viral tumor antigens were explored and shown to have common features and mechanisms even though they appear to have evolved from diverse genes. The SV40 large tumor antigen, the human Papilloma virus E7 protein and the Adenovirus E1A protein were shown to bind to and inactivate the functions of the Retinoblastoma proteins in transformed cells. This resulted in the activation of the E2F and DP transcription factors and the entry of cells into the S-phase of DNA synthesis which was required for viral DNA replication. These events triggered the activation of p53 which promotes apoptosis of these virus infected cells limiting virus replication and tumor formation. These viruses responded by evolving and producing the SV40 large tumor antigen, the human Papilloma virus E6 protein and the Adenovirus E1b-55Kd protein which binds to and inactivates the p53 functions in both the infected cells and transformed cells. Some of the human Papilloma viruses and one of the Polyoma viruses have been shown to cause selected cancers in humans. Both the p53 tumor suppressor gene, which was uncovered in the studies with these viruses, and the retinoblastoma protein, have been shown to play a central role in the origins of human cancers via both somatic and germ line mutations in those genes.

Solubility survey of fragments of the neurofibromatosis type 1 protein neurofibromin

The protein giant neurofibromin (320 kDa) is the protein product of the NF1 tumor suppressor gene, alterations of which are responsible for the pathogenesis of neurofibromatosis type 1 (NF1). Neurofibromin is a Ras-specific GTPase activating protein (RasGAP) that, 15 years after the cloning of the gene, remains the only clearly defined function of the protein. In a structural proteomics approach, we aimed at defining functions beyond RasGAP activity based on the discovery of structural modules. Given the poor outcome of domain prediction tools, we have undertaken a fragment solubility survey covering the full protein sequence, with the aim of defining new domain boundaries or fragments that could be investigated by biochemical methods including structural analysis. More than 200 constructs have been expressed and tested for solubility in small scale assays. Boundaries were chosen based upon secondary structure predictions, sequence conservation among neurofibromin orthologues and chemical properties of amino acids. Using this strategy we recently discovered a novel bipartite module in neurofibromin. We have expanded our study to include ESPRIT, a library-based construct screen, to perform fragment testing at a finer level with respect to the choice of terminal residues. Our study confirms earlier notions about the challenges neurofibromin presents to the biochemist and points to strategies whereby the success rate may be enhanced in the future.

Recent insights into the molecular mechanisms involved in aging and the malignant transformation of adult stem/progenitor cells and their therapeutic implications

Recent advancements in tissue-resident adult stem/progenitor cell research have revealed that enhanced telomere attrition, oxidative stress, ultraviolet radiation exposure and oncogenic events leading to severe DNA damages and genomic instability may occur in these immature and regenerative cells during chronological aging. Particularly, the alterations in key signaling components controlling their self-renewal capacity and an up-regulation of tumor suppressor gene products such as p16 INK4A, p19 ARF, ataxia-telangiectasia mutated (ATM) kinase, p53 and/or the forkhead box O (FOXOs) family of transcription factors may result in their dysfunctions, growth arrest and senescence or apoptotic death during the aging process. These molecular events may culminate in a progressive decline in the regenerative functions and the number of tissue-resident adult stem/progenitor cells, and age-related disease development. Conversely, the telomerase re-activation and accumulation of numerous genetic and/or epigenetic alterations in adult stem/progenitor cells with advancing age may result in their immortalization and malignant transformation into highly leukemic or tumorigenic cancer-initiating cells and cancer initiation. Therefore, the cell-replacement and gene therapies and molecular targeting of aged and dysfunctional adult stem/progenitor cells including their malignant counterpart, cancer-initiating cells, hold great promise for treating and even curing diverse devastating human diseases. These diseases include premature aging diseases, hematopoietic, cardiovascular, musculoskeletal, pulmonary, ocular, urogenital, neurodegenerative and skin disorders and aggressive and recurrent cancers.

The Menin Tumor Suppressor Functions as a Molecular Adapter That Tethers LEDGF to MLL Proteins in Leukemogenesis.

MLL gene rearrangements are present in 5 to 10% of acute leukemias, which are generally associated with a poor prognosis. Chromosomal translocations at the MLL locus create MLL fusion genes that constitute 5‘ portions of MLL and 3‘ portions of partner genes. The resultant MLL fusion proteins transform myeloid progenitors by inappropriately activating a HOX-associated self renewal program. However, the molecular mechanisms underlying MLL oncoprotein function are not fully understood. Previously we identified menin as a component of the MLL macromolecular complex. Menin is a product of the MEN1 tumor suppressor gene, whose loss of function causes multiple endocrine neoplasia type 1 (MEN1). Despite its tumor suppressor role in endocrine tissues, menin functions as an essential oncogenic cofactor for MLL fusion protein-dependent leukemogenesis. This notion was supported by three lines of evidence:MLL fusion proteins lacking the menin binding motif do not transform myeloid progenitors;inactivation of menin causes growth arrest and subsequent differentiation of MLL oncogene transformed cells; andmenin is required for MLL oncogene dependent transcriptional activation of HOX genes.These findings raised a fundamental question: how does menin contribute to MLL-dependent transcription? Because menin lacks known functional motifs, its molecular functions could not be deduced from its structure. We hypothesized that menin may function as an adapter that tethers MLL to unknown associated factors. To identify such associated factors, we performed affinity purification of the MLL-ENL/menin complex from nuclear extracts of cells that transiently over-expressed both MLL-ENL and menin. Mass spectrometry identified LEDGF, originally identified as a transcriptional co-activator, in the purified material as a novel associated factor. LEDGF associates conjointly with the MLL/menin complex but not with MLL or menin alone, supporting the hypothesis that menin plays an adapter role to bridge MLL and LEDGF. Further analysis revealed that LEDGF is critical for MLL fusion protein-dependent leukemogenesis. Fine mapping of the domain responsible for LEDGF binding determined MLL residues 109–153 as the LEDGF binding domain (LBD). Mutations in LBD resulted in loss of oncogenic activity of MLL fusion proteins. Moreover, knock down of LEDGF in MLL-transformed cells caused growth arrest and differentiation in the same manner as menin knock down. These results demonstrate that LEDGF is also required for the initiation and maintenance of MLL fusion protein-dependent transformation. In contrast to menin, LEDGF has a distinctive functional motif (the PWWP domain), which reportedly has chromatin binding activity. To further confirm that menin is an adapter that links MLL and LEDGF, we examined the oncogenic functions of an artificial MLL fusion protein whose menin binding motif is replaced by the PWWP domain of LEDGF. This PWWP-MLL-ENL fusion protein does not associate with menin because it lacks the menin binding motif, nevertheless transforms myeloid progenitors. Chromatin immunoprecipitation experiments show that the PWWP-MLL- ENL fusion protein localizes at the HOXA9 locus while menin is absent. Moreover, myeloid progenitors transformed by the PWWP-MLL-ENL fusion protein continue to proliferate after menin is genetically inactivated. Thus covalent tethering of the PWWP domain fully compensates for loss of menin's cofactor function. Therefore, menin's only role in MLL-associated leukemogenesis is to tether LEDGF to MLL fusion proteins. In summary, this study identifies a previously unknown essential oncogenic cofactor of MLL fusion proteins and proposes a stepwise association model in which the MLL fusion protein first associates with menin, then recruits LEDGF to its complex to become functionally active.

The Neurofibromatosis 2 Tumor Suppressor Gene Product, Merlin, Regulates Human Meningioma Cell Growth by Signaling through YAP

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the occurrence of schwannomas and meningiomas. Several studies have examined the ability of the NF2 gene product, merlin, to function as a tumor suppressor in diverse cell types; however, little is known about merlin growth regulation in meningiomas. In Drosophila, merlin controls cell proliferation and apoptosis by signaling through the Hippo pathway to inhibit the function of the transcriptional coactivator Yorkie. The Hippo pathway is conserved in mammals. On the basis of these observations, we developed human meningioma cell lines matched for merlin expression to evaluate merlin growth regulation and investigate the relationship between NF2 status and Yes-associated protein (YAP), the mammalian homolog of Yorkie. NF2 loss in meningioma cells was associated with loss of contact-dependent growth inhibition, enhanced anchorage-independent growth and increased cell proliferation due to increased S-phase entry. In addition, merlin loss in both meningioma cell lines and primary tumors resulted in increased YAP expression and nuclear localization. Finally, siRNA-mediated reduction of YAP in NF2-deficient meningioma cells rescued the effects of merlin loss on cell proliferation and S-phase entry. Collectively, these results represent the first demonstration that merlin regulates cell growth in human cancer cells by suppressing YAP.

Apoptotic signaling by c-MYC

c-MYC has a pivotal function in growth control, differentiation and apoptosis, and its abnormal expression is associated with many tumors. Overexpression of c-MYC sensitizes cells to apoptosis by a variety of stimuli. The decision of a cell to undergo apoptosis and how this apoptotic response is regulated by c-MYC depends on the specific cell type and the physiological status of the cell. Multiple cooperating molecular pathways of cell survival and apoptosis determine whether a cell lives or dies, and understanding how c-MYC interfaces with these pathways to influence the survival of cells is important to understand normal and abnormal development, tumor initiation and progression, and response of tumors to different treatment regimens. This article will provide an overview of the function of the tumor suppressor gene product p53 in the c-MYC-mediated apoptotic response and how c-MYC amplifies the intrinsic mitochondrial pathway and triggers and/or amplifies the death receptor pathways. Finally, a model for how deregulated c-MYC prematurely triggers the normal apoptotic response associated with terminal myeloid differentiation while also blocking the differentiation program is presented.

Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G1 cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (1/5; 20%) and pancreatic (3/19; 15.7%) carcinoma lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53/DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

COOH-Terminal Binding Protein Regulates Expression of the p16INK4A Tumor Suppressor and Senescence in Primary Human Cells

The p16/pocket-protein pathway sets a balance between tumor suppression and capacity for tissue regeneration. Understanding the upstream signaling pathway that turns on the expression of p16 is required both for knowing the tumorigenic stresses from which this pathway provides protection and for appreciating the selective pressure that leads to the loss of this pathway in most human tumors. We report that COOH-terminal binding protein (CtBP), a physiologically regulated transcriptional corepressor that dimerizes to hold together repressive complexes, regulates p16 expression in primary human fibroblasts and keratinocytes. Interfering with CtBP-mediated repression increased p16 expression and accelerated senescence. CtBP had little influence on the expression of the alternate product of the CDKN2A tumor-suppressor gene, p14(ARF). Loss of CtBP-mediated repression diminished the Polycomb-based epigenetic histone mark that is reported to favor silencing of p16 via DNA methylation. Enhancing CtBP-mediated repression by growing cells in low oxygen increased the association of CtBP with the p16 promoter, as assessed by chromatin immunoprecipitation, and reduced p16 expression. Stresses and stimuli that reduce CtBP-mediated repression are associated with increased p16 expression; therefore, CtBP may provide a common final target for regulating the balance among tumor suppression, regenerative capacity, and senescence.

Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers

Telomerase activation is a critical step for human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. Transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is the major mechanism for cancer-specific activation of telomerase, and a number of factors have been identified to directly or indirectly regulate the hTERT promoter, including cellular transcriptional activators (c-Myc, Sp1, HIF-1, AP2, ER, Ets, etc.) as well as the repressors, most of which comprise tumor suppressor gene products, such as p53, WT1, and Menin. Nevertheless, none of them can clearly account for the cancer specificity of hTERT expression. The chromatin structure via the DNA methylation or modulation of nucleosome histones has recently been suggested to be important for regulation of the hTERT promoter. DNA unmethylation or histone methylation around the transcription start site of the hTERT promoter triggers the recruitment of histone acetyltransferase (HAT) activity, allowing hTERT transcription. These facts prompted us to apply these regulatory mechanisms to cancer diagnostics and therapeutics. Telomerase-specific replicative adenovirus (Telomelysin, OBP-301), in which E1A and E1B genes are driven by the hTERT promoter, has been developed as an oncolytic virus that replicates specifically in cancer cells and causes cell death via viral toxicity. Direct administration of Telomelysin was proved to effectively eradicate solid tumors in vivo, without apparent adverse effects. Clinical trials using Telomelysin for cancer patients with progressive stages are currently ongoing. Furthermore, we incorporated green fluorescent protein gene (GFP) into Telomelysin (TelomeScan, OBP-401). Administration of TelomeScan into the primary tumor enabled the visualization of cancer cells under the cooled charged-coupled device (CCD) camera, not only in primary tumors but also the metastatic foci. This technology can be applied to intraoperative imaging of metastatic lymphnodes. Thus, we found novel tools for cancer diagnostics and therapeutics by utilizing the hTERT promoter.

Regulation of Bax activation and apoptotic response to UV irradiation by p53 transcription-dependent and -independent pathways

The Trp53 tumor suppressor gene product (p53) functions in the nucleus to regulate proapoptotic genes, whereas cytoplasmic p53 directly activates proapoptotic Bcl-2 proteins to permeabilize mitochondria and initiate apoptosis. Here, we demonstrate that both p53 transcription-dependent and -independent pathways contribute to UV-induced apoptosis. First we show that Pifithrin-α, a small molecule inhibitor of p53 transcriptional activity, delays Bax translocation and cell death by UV irradiation. Then using CHX (cycloheximide) to prevent new protein expression in response to p53, we also find that Bax translocation and cell death by UV irradiation are delayed. Furthermore we find that overexpression of Bcl-x L, an inhibitor of cytoplasmic p53 after UV irradiation, prevents cell death. Finally, we observe that Pifithrin-α and CHX effectively inhibit PUMA expression by UV irradiation. Taken together, these data indicate that the nuclear p53 promotes PUMA expression, which then displaces cytoplasmic p53 from Bcl-x L, allowing p53 to induce mitochondrial permeabilization, thereby triggering apoptosis.

Skp2 inversely correlates with p27 and tuberin in transformed cells

The cyclin-dependent kinase inhibitor p27Kip1 (p27) is a major gatekeeper of the mammalian cell cycle progression known to be regulated by both, its subcellular localization and its degradation. To allow entrance into S phase and thereby mammalian cell cycle progression p27 must be degraded by a skp2-containing E3 ubiquitin ligase whose task is to target p27 for degradation by the proteasome. The tumor suppressor gene product tuberin directly binds to p27 and protects it from degradation via skp2. Whereas, p27 and tuberin are known to be localized to both, the cytoplasm and the nucleus, the localization of skp2 remained elusive. Here we demonstrate that skp2 is a cytoplasmic and nuclear protein. In addition we found an inverse correlation of the endogenous protein levels of skp2 with p27 and tuberin in different transformed cells and under different growth conditions. These data allow new important insights into this molecular network of cell cycle control.

Down‐regulation of claudin‐2 in breast carcinomas is associated with advanced disease

Claudin 2 (CLDN2) is a family of integral membrane tight junctions. The aim was to determine the influence of CLDN2 expression on tumour behaviour and its role in breast carcinogenesis. Thirty-seven invasive breast carcinomas and corresponding normal breast tissues were examined for CLDN2 protein and mRNA expression using Western blotting and semiquantitative reverse transcriptase-polymerase chain reaction. The expression of CLDN2 protein in 118 cases of breast carcinoma was further studied with immunohistochemistry and related to various clinicopathological parameters. CLDN2 protein expression was significantly down-regulated (0.4-fold) in tumours compared with corresponding normal breast tissue (P < 0.0001). Down-regulation of CLDN2 was significantly associated with lymph node metastasis (P = 0.047) by Western blot analysis, and with high clinical stage (P = 0.040) by immunohistochemistry. The expression levels of CLDN2 mRNA in high clinical stages (stages II and III) were lower than those in low clinical stage (stage I) and normal tissue, but not statistically significantly so. These results suggest that CLDN2 is implicated in the progression as well as the development of breast carcinoma, indicating that CLDN2 is a possible tumour suppressor gene product.

E2F1 mediates pneumococcal-induced brain damage

Understanding the signaling pathways of stress-induced cell death is important for the development of new treatment regimens for neurodegenerative diseases. In acute pneumococcal meningitis, cell death in the hippocampus is mediated in part by the serine/threonine kinase Atm and the tumor suppressor gene product p53 (Mitchell et al. in J Infect Dis 190:3039–3046, 2004) followed by caspase-3 activation (Braun et al. in Nat Med 5:298–302, 1999). p53 communicates also with the transcriptional cell cycle regulator E2F1; for instance, p53-induced cell death can be accelerated by co-transfection of E2F1 (Wu and Levine in Proc Natl Acad Sci USA 91:3602–3606, 1994). Although members of the E2F transcription factor family are primarily required for propagation of the cell cycle, E2F1 is also involved in mediating apoptotic programs both by p53dependent and p53-independent pathways (Stanelle and Putzer in Trends Mol Med 12:177–185, 2006). For instance, experimental cerebral infarcts are decreased in E2F1 knockout mice (MacManus et al. in Neuroreport 10:2711–2714, 1999). The aim of this study was to investigate the role of E2F1 in the induction of neuronal damage in pneumococcal-induced meningitis. E2F1 knockout mice were on a C57BL/6 J background and backcrossed at least ten times with the C57BL/6 J mouse strain. Following anesthesia with ketamine (35 mg/ kg) and xylazine (5 mg/kg), bacterial meningitis was induced in C57BL/6 J wild-type (n = 4) and E2F1i/i mice (Field et al. in Cell 85:549–561, 1996) (n = 4) (»20 g body weight) by lumbar injection of Streptococcus pneumoniae (10 CFU of encapsulated serotype 2 pneumococcal strain D39 in 30 l PBS) (Braun et al. in Nat Med 5:298–302, 1999). To quantify bacterial growth 20 h later, CSF was taken from the cisterna magna and cultured on blood agar plates. Mice were perfused transcardially and brains were embedded in paraYn. To detect pycnotic nuclei, sections were stained with hematoxylin and eosin (HE johann.braun@charite.de

Mitochondrial p32 Is a Critical Mediator of ARF-Induced Apoptosis

The shared exon 2 of the p14ARF-p16INK4a locus is frequently mutated in human cancers. However, in contrast to the exon 1beta-encoded N-terminal half of ARF, the function of the exon 2-encoded C-terminal half of ARF has been elusive. Here, we report that the mitochondrial protein p32/C1QBP binds the ARF C terminus. We show that p32 is required for ARF to localize to mitochondria and induce apoptosis, and that ARF mutations specifically disrupting p32 binding can impair both of these functions. Wild-type ARF, but not a p32-binding-deficient ARF mutant, localizes to mitochondria, reduces mitochondrial membrane potential, and sensitizes cells to p53-induced apoptosis. These findings provide a potential explanation for the frequent human cancer mutations targeting the ARF C terminus.

Tumor risk assessment by means of immunocytochemical detection of early pre-malignant changes in buccal smears

Increasing incidence of carcinomas in the upper aero-digestive tract, both in Germany and in other European countries requires development of new preventive strategies. The cure rate at advanced tumor stages remains poor in spite of a variety of available therapeutic methods. In the present study the quantitative assessment of a pre-malignant mucosa lesion within a field cancerization was performed by means of immunocytochemical methods. This may allow individuals with an increased risk of developing malignant disease to be identified. Cytosmears taken from healthy buccal mucosa of tumor patients (n=50) and from healthy probands (n=100) with different tobacco and alcohol consumption levels were examined with regard to identifying increased expression of the proliferation markers (PCNA, MIB1), of the tumor suppressor gene product p53 as well as the oncogene product cyclin D1. There was a significant difference in expression of investigated proliferation markers between tumor patients and healthy probands (p<0.0001). When comparing the rate of positively marked cell nuclei to cigarette pack years the marker cyclin D1 and MIB1 show an increased rate in the groups with high tobacco consumption as compared to the group with a low exposure (p>0.05). It could be possible to use the marker MIB1 and cyclin D1 to screen risk groups, since the relative morbidity risk (odds ratio) increases (by 45-62 times) if the threshold value of 4 positively marked cell nuclei is exceeded.