E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry.

The E2F transcription factor couples the coordinate expression of cell cycle proteins to their appropriate transition points. Its activity is controlled by the cell cycle regulators pRB, p107, and p130. These bind to E2F at defined but distinct stages of the cell cycle. Using specific antisera, we have identified the DP and E2F components of each of these species. Although present at very different levels, DP-1 and DP-2 are evenly distributed among each of these complexes. In contrast, the individual E2Fs have distinctly different binding profiles. Consistent with previous studies, E2F-1, E2F-2, and E2F-3 bind specifically to the retinoblastoma protein. In each case, their expression and DNA binding activity are restricted to post-G1/S fractions. Surprisingly, E2F-1 and E2F-3 make unequal contributions to the pRB-associated and free E2F activity, suggesting that these proteins perform different cell cycle functions. Most significantly, this study showed E2F-4 accounts for the vast majority of the endogenous E2F activity. In arrested cells, E2F-4 is sequestered by the p130 protein. However, as the cells pass the G1-to-S transition, the levels of pRB and p107 increase and E2F-4 now associates with both of these regulators. Despite this, a considerable amount of E2F-4 exists as free E2F. In G1 cells, this accounts for almost all of the free activity. Once the cells enter S phase, free E2F is composed of an equal mixture of E2F-4 and E2F-1.

Specific regulation of E2F family members by cyclin-dependent kinases.

The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases.

In vivo association of E2F and DP family proteins.

The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle. This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein (pRB) or its relatives p107 and p103. The first cDNA from the E2F family to be cloned was designated E2F-1, and multiple E2F family members have now been identified. They bind to DNA as heterodimers, interacting with proteins known as DP. Here we demonstrate that DP is also a family of polypeptides with at least two members (hDP-1 and hDP-2). Both hDP-1 and hDP-2 bind to all E2F family members in vivo, and each complex is capable of activating transcription. However, the various E2F/DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo, and the specificity of pRB or p107 binding is mediated by the E2F subunit.

The retinoblastoma protein physically associates with the human cdc2 kinase.

The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle.

A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor.

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.

E2F4 is exported from the nucleus in a CRM1-dependent manner.

E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G1. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16(INK4a), an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G1 in association with a suitable pocket protein.

E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

The retinoblastoma protein binds to a family of E2F transcription factors.

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.

Benzalkonium tolerance genes and outcome in Listeria monocytogenes meningitis.

OBJECTIVES: Listeria monocytogenes is a food-borne pathogen that can cause meningitis. The listerial genotype ST6 has been linked to increasing rates of unfavourable outcome over time. We investigated listerial genetic variation and the relation with clinical outcome in meningitis. METHODS: We sequenced 96 isolates from adults with listerial meningitis included in two prospective nationwide cohort studies by whole genome sequencing, and evaluated associations between bacterial genetic variation and clinical outcome. We validated these results by screening listerial genotypes of 445 cerebrospinal fluid and blood isolates from patients over a 30-year period from the Dutch national surveillance cohort. RESULTS: We identified a bacteriophage, phiLMST6 co-occurring with a novel plasmid, pLMST6, in ST6 isolates to be associated with unfavourable outcome in patients (p 2.83e-05). The plasmid carries a benzalkonium chloride tolerance gene, emrC, conferring decreased susceptibility to disinfectants used in the food-processing industry. Isolates harbouring emrC were growth inhibited at higher levels of benzalkonium chloride (median 60 mg/L versus 15 mg/L; p <0.001), and had higher MICs for amoxicillin and gentamicin compared with isolates without emrC (both p <0.001). Transformation of pLMST6 into naive strains led to benzalkonium chloride tolerance and higher MICs for gentamicin. CONCLUSIONS: These results show that a novel plasmid, carrying the efflux transporter emrC, is associated with increased incidence of ST6 listerial meningitis in the Netherlands. Suggesting increased disease severity, our findings warrant consideration of disinfectants used in the food-processing industry that select for resistance mechanisms and may, inadvertently, lead to increased risk of poor disease outcome.

Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.

BACKGROUND: PTEN tumor suppressor gene mutations are the most frequent genetic lesions in endometrial adenocarcinomas of the endometrioid subtype. Testing the hypothesis that altered PTEN function precedes the appearance of endometrial adenocarcinoma has been difficult, however, partly because of uncertainties in precancer diagnosis. METHODS: Two series of endometrial cancer and precancer (endometrial intraepithelial neoplasia, as diagnosed by computerized morphometric analysis) tissue samples were studied, one for PTEN mutations by the use of denaturing gradient gel electrophoresis and another for PTEN protein expression by immunohistochemistry. Endometria altered by high estrogen levels that are unopposed by progestins-conditions known to increase cancer risk-were also studied by immunohistochemistry. Fisher's exact test was used for statistical analysis. RESULTS: The PTEN mutation rate was 83% (25 of 30) in endometrioid endometrial adenocarcinomas and 55% (16 of 29) in precancers, and the difference in number of mutations was statistically significant (two-sided P =.025). No normal endometria showed PTEN mutations. Although most precancers and cancers had a mutation in only one PTEN allele, endometrioid endometrial adenocarcinomas showed complete loss of PTEN protein expression in 61% (20 of 33) of cases, and 97% (32 of 33) showed at least some diminution in expression. Cancers and most precancers exhibited contiguous groups of PTEN-negative glands, while endometria altered by unopposed estrogens showed isolated PTEN-negative glands. CONCLUSIONS: Loss of PTEN function by mutational or other mechanisms is an early event in endometrial tumorigenesis that may occur in response to known endocrine risk factors and offers an informative immunohistochemical biomarker for premalignant disease. Individual PTEN-negative glands in estrogen-exposed endometria are the earliest recognizable stage of endometrial carcinogenesis. Proliferation into dense clusters that form discrete premalignant lesions follows.

PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death.

PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.

Structural organization and expression of the mouse estrogen receptor.

Complementary DNA clones corresponding to the mouse uterus estrogen receptor mRNA have been isolated and characterized. Nucleotide sequence analysis predicts that full-length cDNA has the potential to code for a polypeptide of 599 amino acids, and comparison with the protein sequences of the rat, human, and chicken estrogen receptors reveals overall homologies of 97%, 88% and 77%, respectively. Genomic clones for the mouse estrogen receptor have been isolated from a cosmid library and used in conjunction with the cDNA clones to study the expression of the receptor in vivo by RNase mapping, primer extension, and Northern blotting. These analyses demonstrate that transcription initiates at multiple sites which span a region of at least 62 base pairs and that the estrogen receptor is encoded by mRNA of approximately 6.5 kilobases in size. There are 10 major starts in total, one of which is situated 31 nucleotides downstream from a TATA box-like motif and coincides with the start of the cDNA clone pMOR8. The ability of the cDNA clone to produce a functional protein was verified by transfection into COS-1 cells which lack endogenous estrogen receptor. The mouse estrogen receptor, in a SV40-based expression vector, was cotransfected with a chimeric marker plasmid consisting of an estrogen response element from the vitellogenin A2 gene linked to the thymidine kinase promoter and the chloramphenicol acetyl transferase gene. In the presence of estradiol chloramphenicol acetyl transferase activity is stimulated by up to 80-fold, while tamoxifen and 4-hydroxytamoxifen act primarily as antiestrogens in this in vitro assay.

A proposed consensus steroid-binding sequence--a reply.

A consensus sequence has been proposed as necessary for steroid binding on the basis of sequence homology in steroidogenic enzymes, steroid binding proteins and steroid receptors. We have mapped the limit of sequences important for steroid binding in the mouse estrogen receptor using Scatchard analysis of deletion mutants. Since a mutant lacking the entire proposed consensus is still able to bind estradiol with essentially wild type affinity, the functional significance of this motif is unclear.

Inactivation of the retinoblastoma gene yields a mouse model of malignant colorectal cancer.

The retinoblastoma gene (Rb) is mutated at significant frequency in various human epithelial tumors, including colorectal cancer, and is strongly associated with metastatic disease. However, sole inactivation of Rb in the mouse has so far failed to yield epithelial cancers. Here, we specifically inactivate Rb and/or p53 in the urogenital epithelium and the intestine. We find that the loss of both tumor suppressors is unable to yield tumors in the transitional epithelium lining the bladder, kidneys and ureters. Instead, these mice develop highly metastatic tumors of neuroendocrine, not epithelial, origin within the urogenital tract to give prostate cancer in the males and vaginal tumors in the females. Additionally, we discovered that the sole inactivation of Rb in the intestine was sufficient to induce formation of metastatic colorectal adenocarcinomas. These tumors closely mirror the human disease in regard to the age of onset, histological appearance, invasiveness and metastatic potential. Like most human colorectal carcinomas, our murine Rb-deficient tumors demonstrate genomic instability and they show activation of ß-catenin. Deregulation of the Wnt/ß-catenin pathway is specific to the intestinal tumors, as genomic instability but not activation of ß-catenin was observed in the neuroendocrine tumors. To date, attempts to generate genetically engineered mouse models of colorectal cancer tumors have yielded mostly cancer of the small intestine, which rarely occurs in humans. Our system provides the opportunity to accurately model and study colorectal cancer in the mouse via a single gene mutation.

Surgical treatment of severe autistic regression in childhood epilepsy.

We report 2 children with partial epilepsy who manifested social and language regression and partial recovery after surgical treatment. One child had seizures since the first 2 weeks of life, caused by a right temporal dysembryoplastic neuroepithelial tumor and regression in the latter part of the first year; seizures were relieved and some functions were recovered after temporal lobe resection at 12 months of age. The second child developed epilepsy at 3 years 3 months, and between 5 years 9 months and 6 years 1 month he became aphasic (Landau-Kleffner syndrome) and lost social functioning, manifesting a very severe behavior disorder. He exhibited a significant improvement in communication, social functioning, and behavior after left multiple subpial transections. Both children manifested evidence of subclinical seizure activity in both temporal lobes. Their clinical picture was one of combined language and autistic regression, and the autistic features demonstrated a clear response to surgical treatment. We suggest that in pediatric epilepsy surgical programs, autistic regression should prompt urgent investigation if drug treatment is not effective.

Floodplain lakes as sinks for sediment-associated contaminants--a new source of proxy hydrological data?

Lake and reservoir sediments often contain valuable records of sediment yield history and sediment-associated nutrient and contaminant transport spanning timescales from decades to millennia. Nevertheless, there have been few attempts to evaluate floodplain lakes as a source of proxy hydrological data for reconstructing short-term trends in sediment-associated nutrient and contaminant transport. Results from an analysis of floodplain lake sediment cores suggested good preservation of the 137Cs record, which provided an absolute dating control. Changes in the concentration of sediment-associated heavy metals and phosphorus were observed downcore and the analysis of mineral magnetic properties and particle size were used to identify the influx of sediment associated with high magnitude, low frequency flood events. Although floodplain lake-sediments only preserve a partial record, they may provide a valuable source of proxy hydrological data for reconstructing trends in sediment and sediment-associated contaminant transport where long-term sediment monitoring programmes are not available.

Particulate phosphorus transport by sub-surface drainage from agricultural land in the UK. Environmental significance at the catchment and national scale.

Phosphorus (P) is the key limiting nutrient in most UK freshwater systems. With increased legislation controlling point source inputs, dissolved (DP) and particulate P (PP) derived from diffuse sources are making a more significant contribution to the total P loading of surface waters. Recent research has focused on pathways linking diffuse sources to the fluvial system and sub-surface field drains have been shown to transport both sediment and P rapidly to watercourses. Preliminary results are presented from an ongoing study using environmental tracers to identify the source of the drain sediment and its potential as a carrier of PP. These results suggest that the majority of sediment in drains is topsoil derived, but the significance of P loss via this pathway in a regional or UK context has yet to be evaluated. A protocol to study the potential problem at a regional/national scale is discussed and initial data presented.

Particulate-associated heavy metals in the urban environment: their transport from source to deposit, Coventry, UK.

A sequential digest was used to examine the speciation of particulate-associated heavy metal pollutants in a holistic approach to the study of the movement of sediment within the urban environment. Sediments were classified according to whether they mainly acted as sources, were mostly transported, or had become deposited and were fractionated into two particle sizes to reflect sediment deposited in lakes (< 63 microns) and transported in rivers (< 2 mm). Results showed that the two particle size fractions yielded the same data, but that trends were found in terms of dominant heavy metal species as the sediment moved through the source-transport-deposit cascade. Whilst the results highlighted the complexity of the urban environment, a sequential digestion of the samples enabled comments to be made in terms of risk which could subsequently feed in to a management strategy for polluted urban sediments.

Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer.

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1(-/-) mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1(-/-) adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.

Loss of pRB and p107 disrupts cartilage development and promotes enchondroma formation.

The pocket proteins pRB, p107 and p130 have established roles in regulating the cell cycle through the control of E2F activity. The pocket proteins regulate differentiation of a number of tissues in both cell cycle-dependent and -independent manners. Prior studies showed that mutation of p107 and p130 in the mouse leads to defects in cartilage development during endochondral ossification, the process by which long bones form. Despite evidence of a role for pRB in osteoblast differentiation, it is unknown whether it functions during cartilage development. Here, we show that mutation of Rb in the early mesenchyme of p107-mutant mice results in severe cartilage defects in the growth plates of long bones. This is attributable to inappropriate chondrocyte proliferation that persists after birth and leads to the formation of enchondromas in the growth plates as early as 8 weeks of age. Genetic crosses show that development of these tumorigenic lesions is E2f3 dependent. These results reveal an overlapping role for pRB and p107 in cartilage development, endochondral ossification and enchondroma formation that reflects their coordination of cell-cycle exit at appropriate developmental stages.