1: Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7331-6. Epub 2003 May 30. Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors. Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, Lin SM, Wickramasinghe R, Scott MP, Wechsler-Reya RJ. Department of Pharmacology and Cancer Biology and Bioinformatics Shared Resource, Duke University Medical Center, Durham, NC 27710, USA. Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. We used DNA microarrays to identify targets of Shh in these cells and found that Shh activates a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis. PMID: 12777630 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Nat Genet. 2003 Jun;34(2):226-30. Gene expression phenotypic models that predict the activity of oncogenic pathways. Huang E, Ishida S, Pittman J, Dressman H, Bild A, Kloos M, D'Amico M, Pestell RG, West M, Nevins JR. Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina 27710, USA. High-density DNA microarrays measure expression of large numbers of genes in one assay. The ability to find underlying structure in complex gene expression data sets and rigorously test association of that structure with biological conditions is essential to developing multi-faceted views of the gene activity that defines cellular phenotype. We sought to connect features of gene expression data with biological hypotheses by integrating 'metagene' patterns from DNA microarray experiments in the characterization and prediction of oncogenic phenotypes. We applied these techniques to the analysis of regulatory pathways controlled by the genes HRAS (Harvey rat sarcoma viral oncogene homolog), MYC (myelocytomatosis viral oncogene homolog) and E2F1, E2F2 and E2F3 (encoding E2F transcription factors 1, 2 and 3, respectively). The phenotypic models accurately predict the activity of these pathways in the context of normal cell proliferation. Moreover, the metagene models trained with gene expression patterns evoked by ectopic production of Myc or Ras proteins in primary tissue culture cells properly predict the activity of in vivo tumor models that result from deregulation of the MYC or HRAS pathways. We conclude that these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state, whether in vitro or in vivo, in a way that truly reflects the complexity of the regulatory pathways that are affected. PMID: 12754511 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Mol Cell. 2001 Jul;8(1):105-13. Myc requires distinct E2F activities to induce S phase and apoptosis. Leone G, Sears R, Huang E, Rempel R, Nuckolls F, Park CH, Giangrande P, Wu L, Saavedra HI, Field SJ, Thompson MA, Yang H, Fujiwara Y, Greenberg ME, Orkin S, Smith C, Nevins JR. Division of Human Cancer Genetics, Department of Molecular Virology, Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA. Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions. PMID: 11511364 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: EMBO J. 2000 Nov 1;19(21):5813-23. Induction of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth by Myc are genetically separable events. Beier R, Burgin A, Kiermaier A, Fero M, Karsunky H, Saffrich R, Moroy T, Ansorge W, Roberts J, Eilers M. Institute of Molecular Biology and Tumour Research, Emil-Mannkopff-Strabetae 2, 35033 Marburg, Germany. The c-myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth. We have now used p27(-/-) fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S-phase entry and cell growth without affecting cyclin E-cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27(-/-) MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S-phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E-cdk2 kinase, of E2F-dependent transcription and of cell growth by Myc can be genetically separated from each other. PMID: 11060032 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Oncogene. 2000 Jul 6;19(29):3330-4. A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts. Berns K, Hijmans EM, Koh E, Daley GQ, Bernards R. Division of Molecular Carcinogenesis, and Center for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam. The c-myc gene is frequently over-expressed in human cancers and is involved in regulation of proliferation, differentiation and apoptosis. c-Myc is a transcription factor that acts primarily by regulating the expression of other genes. However, it has been very difficult to identify bona fide c-Myc target genes that explain its diverse biological activities. The recent generation of c-myc deficient Rat1A fibroblasts with a profound and stable growth defect provides a new system to search for genes that can substitute for c-myc in proliferation. In this study, we have attempted to identify genes that rescue the slow growth phenotype of c-myc null cells through introduction of a series of potent cell cycle regulatory genes and several retroviral cDNA expression libraries. None of the candidate genes tested, including SV40 T-antigen and adenovirus E1A, caused reversal of the c-myc null growth defect. Furthermore, extensive screens with high-complexity retroviral cDNA libraries from three different tissue sources revealed that only c-myc and N-myc rescued the c-myc null slow-growth phenotype. Our data support the notion that there are no functional equivalents of the myc family of proto-oncogenes and also suggest that there are no c-Myc-activated genes that alone can substitute for c-Myc in control of cell proliferation. PMID: 10918589 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6481-6. CpG methylation as a mechanism for the regulation of E2F activity. Campanero MR, Armstrong MI, Flemington EK. Department of Cancer Immunology and AIDS, Harvard Medical School and Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA, 02115, USA. Regulation of gene expression in mammals through methylation of cytosine residues at CpG dinucleotides is involved in the development and progression of tumors. Because many genes that are involved in the control of cell proliferation are regulated by members of the E2F family of transcription factors and because some E2F DNA-binding sites are methylated in vivo, we have investigated whether CpG methylation can regulate E2F functions. We show here that methylation of E2F elements derived from the dihydrofolate reductase, E2F1, and cdc2 promoters prevents the binding of all E2F family members tested (E2F1 through E2F5). In contrast, methylation of the E2F elements derived from the c-myc and c-myb promoters minimally affects the binding of E2F2, E2F3, E2F4, and E2F5 but significantly inhibits the binding of E2F1. Consistent with these studies, E2F3, but not E2F1, activates transcription through methylated E2F sites derived from the c-myb and c-myc genes whereas both E2F1 and E2F3 fail to transactivate a reporter gene that is under the control of a methylated dihydrofolate reductase E2F site. Together, these data illustrate a means through which E2F activity can be controlled. PMID: 10823896 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Mol Cell Biol. 2000 May;20(10):3633-9. Complex transcriptional regulatory mechanisms control expression of the E2F3 locus. Adams MR, Sears R, Nuckolls F, Leone G, Nevins JR. Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. E2F transcription activity has been shown to play a critical role in cell growth control, regulating the expression of a variety of genes that encode proteins important for the initiation of DNA replication and cell cycle regulation. We have shown that the E2F3 locus encodes two protein products: the E2F3a product, which is tightly regulated by cell growth, and the E2F3b product, which is constitutively expressed throughout the cell cycle. To further explore the mechanism controlling the expression of the two E2F3 gene products, we analyzed the genomic sequences flanking the 5' region of E2F3a and E2F3b. We find that a series of E2F binding sites confer negative control on the E2F3a promoter in quiescent cells, similar to the control of the E2F1 and E2F2 promoters. In addition, a group of E-box elements, which are Myc binding sites, confer responsiveness to Myc and are necessary for full activation of the E2F3a promoter in response to growth stimulation. Based on these results and past experiments, it appears that the E2F1, E2F2, and E2F3a genes are similarly regulated by growth stimulation, involving a combination of E2F-dependent negative control and Myc-mediated positive control. In contrast, the constitutive expression of the E2F3b gene more closely reflects the control of expression of the E2F4 and E2F5 genes. PMID: 10779353 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Mol Cell. 1999 Feb;3(2):169-79. Ras enhances Myc protein stability. Sears R, Leone G, DeGregori J, Nevins JR. Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. Various experiments have demonstrated a collaborative action of Myc and Ras, both in normal cell growth control as well as during oncogenesis. We now show that Ras enhances the accumulation of Myc activity by stabilizing the Myc protein. Whereas Myc has a very short half-life when produced in the absence of mitogenic signals, due to degradation by the 26S proteasome, the half-life of Myc increases markedly in growth-stimulated cells. This stabilization is dependent on the Ras/Raf/MAPK pathway and is not augmented by proteasome inhibition, suggesting that Ras inhibits the proteasome-dependent degradation of Myc. We propose that one aspect of Myc-Ras collaboration is an ability of Ras to enhance the accumulation of transcriptionally active Myc protein. PMID: 10078200 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Int J Cancer. 1999 Jan 5;80(1):145-54. Frequent deregulation of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma. Diccianni MB, Omura-Minamisawa M, Batova A, Le T, Bridgeman L, Yu AL. Department of Pediatrics/Hematology-Oncology, University of California, San Diego 92103-8447, USA. Alterations of the p16 gene in neuroblastoma are very rare. Pronounced expression of p16 at both the transcript and protein levels, however, was observed in 7 of 19 (39%) neuroblastoma cell lines and 2 of 6 (33%) primary neuroblastoma samples. As p16 expression is tightly controlled in a feedback loop with Rb, we investigated the possibility that changes in p16 expression were reflective of alterations of the downstream components in the G1 regulatory pathway. Two cell lines and one primary sample highly expressing p16 were shown to harbor CDK4 amplification. The cyclin D2 gene was infrequently expressed in neuroblastoma cell lines and did not correlate with p16 expression. Slight variations in the expression of CDK6, cyclins D1, D3 and E; and E2F1 and E2F2 among the cell lines were observed, without apparent correlation with p16 status. No mutations to the p16-binding site of CDK4 and CDK6 nor any mutations to the coding region of p16 itself were identified in neuroblastoma cell lines. Despite frequent N-myc amplification in these cell lines, no relationship with this gene was observed either. All cell lines contained Rb protein with varying degrees of phosphorylation, which bears no correlation with p16 expression. Overall, alterations of the G1 pathway in neuroblastoma included relatively frequent p16 expression and infrequent CDK4 amplification and cyclin D2 expression. Despite a reported feedback relationship between p16 expression and Rb/G1 deregulation, p16 expression in neuroblastoma cell lines is independent of Rb gene and phosphorylation status and, in contrast to other cell lines where expression of p16 leads to G1/S arrest, neuroblastoma cell lines proliferate in the presence of elevated levels of p16. PMID: 9935245 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Pediatr Surg. 1998 Nov;33(11):1695-8. Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma. Komuro H, Valentine MB, Rowe ST, Kidd VJ, Makino S, Brodeur GM, Cohn SL, Look AT. Department of Experimental Oncology, St Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA. BACKGROUND/PURPOSE: Deletion of the short arm of chromosome 1 (1p) is one of the poor prognostic factors in human neuroblastomas. Recent studies have suggested that one or more of the neuroblastoma tumor suppressor genes reside in this region and have identified the shortest region of overlap (SRO) on 1p36. The purpose of this study was to examine deletions of 1p in human neuroblastomas by fluorescence in situ hybridization (FISH). METHODS: Two-color FISH analysis was performed to detect chromosome 1p36 abnormalities in 42 MYCN-amplified neuroblastomas. Four different probes from the 1p36 region, the E2F2, NPPA, D1S160, and CDC2L1 loci were used for detection of 1p abnormalities. A repeat sequence probe, which is specific for the heterochromatic region of chromosome 1 (pUC1.77), was used as a control. RESULTS: Large deletions of 1p36 were observed in 31 (73.8%) of 42 tumors, whereas the remaining 11 (26.2%) showed no deletion. In these 11 tumors, a translocation of 1p was found in one and a duplication of 1p was detected in another. CONCLUSIONS: A strong correlation between 1p abnormalities and MYCN amplification was found in this study. MYCN-amplified neuroblastomas were found to show large deletions of 1p encompassing the SRO. FISH provided a rapid and reliable method to detect hemizygous deletions of 1p. PMID: 9856898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Mol Cell Biol. 1998 Aug;18(8):4565-76. Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein. Buchmann AM, Swaminathan S, Thimmapaya B. Robert H. Lurie Cancer Center and Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, Illinois 60611-3088, USA. The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc, cdc-2, c-neu, and the transforming growth factor beta2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5' upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G1. By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation. PMID: 9671466 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Mol Cell Biol. 1998 Jun;18(6):3620-32. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb. Neuveut C, Low KG, Maldarelli F, Schmitt I, Majone F, Grassmann R, Jeang KT. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0460, USA. Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a). PMID: 9584203 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cancer Res. 1998 Jan 1;58(1):123-34. Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Santoni-Rugiu E, Jensen MR, Thorgeirsson SS. Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892-4255, USA. The oncogene c-myc and transforming growth factor (TGF) alpha are frequently coexpressed in human cancers, suggesting that their interaction may be a critical step in malignant growth. Consistent with this idea, we recently demonstrated in a transgenic mouse model that TGF-alpha dramatically enhances c-myc-induced hepatocarcinogenesis. To elucidate this synergistic effect, we have now investigated regulation of cell cycle and apoptosis during neoplastic development in the liver of c-myc and c-myc/TGFalpha transgenic mice. Both lines displayed dramatic increases of mitotic and apoptotic rates before the onset of hepatocellular carcinoma (HCC), but only c-myc/TGF-alpha livers showed significant levels of net proliferation (mitosis minus apoptosis). Subsequently, mitosis declined in peritumorous tissues, concomitant with the previously reported induction of TGF-beta1, whereas c-myc and c-myc/TGFalpha HCCs maintained mitotic hyperactivity. The c-myc/TGF-alpha HCCs were also characterized by a particularly strong expression of TGF-alpha and very low apoptotic index in contrast to high levels of apoptosis in peritumorous tissues and c-myc HCCs. The differential levels of cell proliferation in noncancerous and cancerous tissues correlated with a stronger induction of cyclin D1 mRNA and protein in c-myc/TGF-alpha and c-myc HCCs associated with intense pRb hyperphosphorylation. Severe deregulation of G1-S transition was also indicated by the dramatic up-regulation, particularly in the HCCs, of pRb-free E2F1-DP1 and E2F2-DP1 transcription factor heterodimers, as assessed by immunoprecipitation and immunohistochemistry. The existence of increased E2F activity during hepatocarcinogenesis was further indicated by the transcriptional induction of putative E2F target genes involved in cell cycle progression, such as endogenous c-myc, cyclin A, Cdc2, and E2F itself. Cdc2 overexpression and the elevated mitotic indices in the HCCs correlated also with induction of cyclin B steady-state levels. The data suggest that coexpression of c-myc and TGF-alpha leads to a selective growth advantage for hepatic (pre)neoplastic cells by disrupting the pRb/E2F pathway and by TGF-alpha-mediated reduction of apoptosis. PMID: 9426068 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Mol Cell Biol. 1997 Sep;17(9):5227-35. Identification of positively and negatively acting elements regulating expression of the E2F2 gene in response to cell growth signals. Sears R, Ohtani K, Nevins JR. Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA. Mammalian cell growth is governed by regulatory activities that include the products of genes such as c-myc and ras that act early in G1, as well as the E2F family of transcription factors that accumulate later in G1 to regulate the expression of genes involved in DNA replication. Previous work has shown that the expression of the E2F1, E2F2, and E2F3 gene products is tightly regulated by cell growth. To further explore the mechanisms controlling accumulation of E2F activity, we have isolated genomic sequences flanking the 5' region of the E2F2 coding sequence. Various assays demonstrate promoter activity in this sequence that reproduces the normal control of E2F2 expression during a growth stimulation. Sequence comparison reveals the presence of a variety of known transcription factor binding sites, including E-box elements that are consensus Myc binding sites, as well as E2F binding sites. We demonstrate that the E-box elements, which we show can function as Myc-responsive sites, contribute in a positive fashion to promoter function. We also find that E2F-dependent negative regulation in quiescent cells plays a significant role in the cell growth-dependent control of the promoter, similar to the regulation of the E2F1 gene promoter. PMID: 9271400 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Cell Growth Differ. 1994 Aug;5(8):789-99. Inhibition of cell growth by TGF beta 1 is associated with inhibition of B-myb and cyclin A in both BALB/MK and Mv1Lu cells. Satterwhite DJ, Aakre ME, Gorska AE, Moses HL. Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. The concept of positive and negative regulation of normal cellular growth by diffusible factors is well illustrated by the effects of epidermal growth factor and transforming growth factor beta 1 (TGF beta 1) on mouse keratinocytes (MK) and mink lung epithelial cells (Mv1Lu). MK and Mv1Lu are nontransformed cell lines that reversibly arrest at a point in late G1 in response to TGF beta 1. Previously, we have shown that expression of the protooncogene c-myc is induced upon epidermal growth factor stimulation of quiescent MK and Mv1Lu cells and that transcriptional suppression of c-myc by TGF beta 1 treatment is important in the TGF beta 1 growth inhibition pathway. Using epidermal growth factor-stimulated synchronized MK and Mv1Lu cells, we have investigated the mRNA expression of a large number of growth factor-inducible genes that are critical regulators of growth in G1 and at the G1/S transition. These genes, often found to be dysregulated in cancer, include transcription factors as well as cyclins and their associated kinases, that promote growth, and tumor suppressor genes, that inhibit growth. As reported here, TGF beta 1 significantly inhibited mRNA expression of B-myb and cyclin A in both cell lines, suggesting that these may be important common downstream targets in the growth inhibition pathway. In contrast, the expression patterns of cyclins D1 and D2 and the transcription factors E2F1 and E2F2 were unaffected in MK cells treated with TGF beta 1 but were significantly inhibited in TGF beta 1-treated Mv1Lu cells. We cite the evidence suggesting that the inhibition of B-myb and cyclin A may contribute to the late G1 arrest caused by TGF beta 1 and that these events may be linked through the actions of the product of the retinoblastoma susceptibility gene (Rb) or an Rb family member. PMID: 7986745 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------