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It is known that at the moment of delivery immediate lost of conceptus (main site of glucose disposal in late pregnancy) is not able to disturb glucose homeostasis in early lactating mothers. However, the mechanism by which this adaptation takes place in early lactation is still unknown. Most studies concerning insulin sensitivity in lactating rats were carried out at 11-13 days postpartum and did not describe functional changes in insulin response in early lactation. Here we show that lactation hypersensitivity to insulin is observed as early as 3 days after delivery (L3). We show that the oxidative soleus muscle displays a transient increased maximal insulin-induced glucose uptake and C[O.sub.2] production, which is temporally limited to L3. Response of soleus muscle was accompanied by an increase in glucose transporter 4 (GLUT4) content at L3. This adaptive response was not detected in the glycolytic plantaris muscle, which displayed lower content of GLUT4. We also found that soleus muscle from early lactating rats have higher insulin receptor expression and tyrosine phosphorylation. Downstream steps of insulin signaling pathway; e.g., insulin receptor substrate 2 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase were also upregulated in soleus muscle. In parallel, protein tyrosine phosphatase 1B expression, a negative regulator of insulin signal, was reduced. Importantly, all of these molecular alterations were time limited to L3 and were not observed in plantaris muscle. These results suggest that improved insulin action in oxidative, but not in glycolytic muscle might contribute to achievement of glucose homeostasis postpartum. soleus muscle; insulin signaling; insulin receptor substrate 2; murine thymoma viral oucogene doi:10.1152/ajpregu.00902.2006

We demonstrate that the susceptibility of human cancer cells to be infected and killed by an oncolytic poxvirus, myxoma virus (MV), is related to the basal level of endogenous phosphorylated Akt. We further demonstrate that nonpermissive tumor cells will switch from resistant to susceptible for MV infection after expression of ectopically active Akt (Myr-Akt) and that permissive cancer cells can be rendered nonpermissive by blocking Akt activation with a dominant-negative inhibitor of Akt. Finally, the activation of Akt by MV involves the formation of a complex between the viral host range ankyrin-repeat protein, M-T5, and Akt. We conclude that the Akt pathway is a key restriction determinant for permissiveness of human cancer cells by MV. oncolytic virus | poxvirus | virus tropism | PkB | M-T5

The eukaryotic translation initiation factor eIF4E is deregulated in many human cancers, and its overexpression in cells leads to malignant transformation. Oncogenic properties of eIF4E are directly linked to its ability to bind 7-methyl guanosine of the 5' mRNA. Here, we observe that the antiviral guanosine analogue ribavirin binds to eIF4E with micromolar affinity at the functional site used by 7-methyl guanosine mRNA cap, competes with eIF4E:mRNA binding, and, at low micromolar concentrations, selectively disrupts eIF4E subcellular organization and transport and translation of mRNAs posttranscriptionally regulated by eIF4E, thereby reducing levels of oncogenes such as cyclin D1. Ribavirin potently suppresses eIF4E-mediated oncogenic transformation of murine cells in vitro, of tumor growth of a mouse model of eIF4E-dependent human squamous cell carcinoma in vivo, and of colony formation of eIF4E-dependent acute myelogenous leukemia cells derived from human patients. These findings describe a specific, potent, and unforeseen mechanism of action of ribavirin. Quantum mechanical and NMR structural studies offer directions for the development of derivatives with improved cytostatic and antiviral properties. In all, ribavirin's association with eIF4E may provide a pharmacologic means for the interruption of post-transcriptional networks of oncogenes that maintain and enhance neoplasia and malignancy in human cancer. cancer | drug | virus | oncogenic network

Senescent chondrocytes accumulate with aging in articular cartilage, a process that interferes with cartilage homeostasis and increases the risk of cartilage degeneration. We showed previously that chondrocyte telomere length declines with donor age, which suggests that the aging process is telomere dependent. From these results we hypothesized that telomerase should delay the onset of senescence in cultured chondrocytes. Population doubling limits (PDL) were determined for chondrocytes expressing telomerase. We found that telomerase alone did not extend PDL beyond controls that senesced after 25 population doublings. The human papillomavirus 16 oncogenes E6 and E7 were transduced into the same cell population to investigate this telomere-independent form of senescence further. Chondrocytes expressing E6 and E7 grew longer than the telomerase cDNA (hTERT) cells but still senesced at 55 population doublings. In contrast, chondrocytes expressing telomerase with E6 and E7 grew vigorously past 100 population doublings. We conclude that although telomerase is necessary for the indefinite extension of chondrocyte life span, telomere-independent senescence limits PDL in vitro and may play a role in the age-related accumulation of senescent chondrocytes in vivo.

Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gin61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5[prime]-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5[prime]-diphosphate-bound inactive Ras and DABP-[P.sub.i]. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-[Beta],[Gamma]-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic [p185.sup.neu]. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-[p185.sup.neu]-treated tumor cells but not in cells expressing internalization defective [p185.sup.neu] receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1,6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.

The Kaposi's sarcoma-associated herpesvirus is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). It is shown that KSHV G-protein-coupled receptor signalling, leads to cell transformation and tumor-igenicity, causing a switch to an angiogenic phenotype. It is concluded that the KSHV G-protein-coupled receptor is a viral oncogene.

Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon-DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone ([E.sub.1]) and estradiol ([E.sub.2]) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-[OHE.sub.1]([E.sub.2])-1([Alpha], [Beta])-N7Gua at 59-213 [[micro]molar]/mol DNA-phosphate whereas the level of stable adducts was 0.1 [[micro]molar]/mol DNA-phosphate. In female Sprague-Dawley rats treated by intramammillary injection of [E.sub.2]-3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 [[micro]molar] 4-[OHE.sub.2]-1([Alpha], [Beta])-N7Gua/molDNA-phosphate. When 4[OHE.sub.1] ([E.sub.2]) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87-440 [[micro]molar] of 4-[OHE.sub.1]([E.sub.2])-1([Alpha], [Beta])-N7Gua was formed. After treatment with 4-[OHE.sub.2], rat mammary tissue contained 1.4 [[micro]molar] of adduct/mol DNA-phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

The retroviral oncogene qin codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the Drosophila Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin-Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin-VP16, failed to transform cells in vitro or in vivo and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.

The interaction of nucleic acids with the p10 Moloney murine leukemia virus (MoMuLV) nucleocapsid protein was analyzed via phosphorescence, fluorescence and optically detected magnetic resonance (OMDR). OMDR analysis of viral p10 proteins purified from MoMuLV viral suspensions indicated the presence of aromatic stacking interactions during the binding of the nucleocapsid protein to poly(I). The high affinity binding of poly(I) with the p10 nucleocapsid protein was also dependent on the native positions of the Tyr and Trp residues in the zinc finger motif.

Viral superantigens, like their bacterial counterpart, induce strong T-cell mediated response and proliferation which is not MHC class II restricted. The murine mammary tumor virus (MMTV) produces a superantigen that stimulates viral transmission to the mammary glands where MMTV produces mammary carcinomas. The murine genomes carrying integrated MMTV proviruses are resistant to infection by MMTV. The superantigen induced T-cell stimulation helps in the transmission of the rabies virus from the gut to the nerve endings.

The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2[K.sup.b] major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility complex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These 'high risk' HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late vital genes, amplification of vital DNA, and production of vital particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative vital life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome ([31E1.sup.*]) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant vital sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late vital gene expression required that the vital genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during vital pathogenesis.

The in vitro reconstituted B region nucleosomes develop in two rotational frames at five discrete translational sites on a 206 bp DNA fragment of the mouse mammary tumor virus. The nucleosome positions are distinguished on the basis of rotational phase obtained from mapping the nucleosome limits with exonuclease III. The positioning signals in the DNA part bears with the energy loss during the DNA-protein contacts.

A study of int-2 transgenic mice infected with mouse mammary tumor virus (MMTV) reveals a new member of the Wnt gene family. This 389-amino acid protein, called Wnt-106, is expressed in the embryo and mammary gland of virgin mice. This gene serves as a candidate oncogene in the mammary epithelium of int-2/Fgf-3 transgenic mice.

Integration of human papillomavirus type 16 DNA genomes elevates steady state levels of mRNAs that code for the viral oncogenes E6 and E7. The increase is attributed to improved stability of E6 and E7 mRNAs from the integrated viral genomes impaired in the 3'untranslated region of the viral early region. The stability of the heterologous mRNA is inhibited by the A+U-rich element in the viral early 3' untranslated region.

A study of polyoma large T antigen (LT) shows the involvement of its 1-260 residue N-terminal (NT) and 264-785 C-terminal (CT) in DNA replication. CT promotes viral DNA replication in growing mouse cells but fails to induce replication in serum-starved cells. NT balances this CT deficiency and initiates S phase. Phosphorylation of CT sites is crucial for replication and it is facilitated by NT.

Studies on new-born chickens suggest that the avian leukemia virus E26 is a single-hit carcinogen and is not a model for two-gene-carcinogenisis. Viral ets may indicate a genetic accident that caused the transduction of proto-ets sequences with proto-myb to generate E26. The E26 deletion mutants exhibit virus filters of about 10(super 5) per ml.

Magnetic resonance spectroscopy is used to verify conformational changes induced by the replacement of bound GDP with GTP analogs. Isotope-edited nuclear magnetic reasonance spectroscopy is used to study the amide resonances of (N-15) glycine and (N-15) isoleucine, or p21(ras)-nucleotide complexes. Active site resonance intensities are attenuated due to the binding of gamma-phosphate of GTP analogs with flexible loops 2 and 4. The substitution of glycine 12 by aspartate alters the sensitivity of the ground state of p21 to nucleotide replacement.

A study of a novel promoter situated upstream from the mouse mammary tumour virus promoter showed that the new U3 promoter enables the manifestation of the superantigen directly when the MMTV promoter is not present, and is active in B lymphocytes, which are cognate antigen-presenting cells for endogenous superantigen. The promoter is located in the endogenous superantigens, encoded by the open reading flame contained within MMTV long terminal repeat (LTR).

Tumorigenesis in transgenic mice carrying the Wnt-1 protooncogene requires the insertion of the mouse mammary tumor virus (MMTV) provirus to impart full tumorigenic potential.The possible genes insertionally activated by the provirus were identified by infecting Wnt-1 mice with MMTV. The results showed that MMTV targets two fibroblast growth factor genes, int-2/Fgf-3 and hst/Fgf-4.

The cellular Jun protein is a trancription factor of the AP-1 complex which is transported in the nucleus via a cell cycle-independent mechanism. However, the oncogenic viral protein (v-Jun) is transported in a cell cyle-dependent manner. An investigation of the nuclear localization signal (NLS) showed that the basic carboxyl-terminal sequence ASKSRKRKL is involved in the cell cycle-dependent nuclear transport of v-Jun. This NLS can effect the transport of Jun-unrelated proteins.

Herpesvirus saimiri is a common cause of T-cell lymphomas in monkeys and is capable of immortalizing monkey T-lymphocytes in vitro. Experiments using lymphocyte cell cultures reveal that the C strain of herpesvirus is also capable of transforming human T-lympphocytes. Results show that infection of human lymphocytes and thymocytes with the group C strain yields immortalized T-cells that express either CD4 of CD8 phenotype. This finding proves that Herpesvirus saimiri may be used to transform human T-cells to produce immortalized lymphocyte cell lines that can be used in immunological and developmental studies.

Nonmetastatic rat mammary cancer (RMC1) cells were fused with highly metastatic RMC1 cells. The highly metastatic cells were produced by transfection of nonmetastatic RMC1with the v-Ha-ras oncogene. The fusant showed the same in vivo tumor growth rate as the highly metastatic parent. However, the metastatic ability was suppressed in the hybrid clones. This suppression of metastatic ability was observed even in the presence of an increased v-Ha-ras p21. These results suggest that the initial aquisition of metastatic ability in the transfected RMC1 was due to the loss of a metastasis suppressor.

Recent research findings have presented evidence that there is a connection between DNA viruses and numerous varieties of human cancer. It has been established that viral agents cause a substantial percentage of malignant diseases throughout the world. Two procedures which would strengthen the link between viruses and cancer are proposed. The first is that there must be a convincing epidemiological association between viruses and cancer. Secondly, the discovery of viral agents in diseased cancer tissue should be accomplished using scientifically competent procedures.

The avian sarcoma virus 16 (ASV 16) is a retrovirus that induces hemangiosarcomas in chickens. Analysis of the ASV 16 genome revealed that it encodes an oncogene that is derived [...]

Overexpression of cyclin D1, an oncogene in tumor-specific DNA rearrangements, in the mammary cells of transgenic mice results in enormous mammary cell production and mammary adenocarcinoma growth. Overexpression leads to deregulation of cell growth and initiation of tumorigenic effects in mammary tissues. The cyclin D1 along with other oncogenic lesions is vital in the induction of malignant breast cancer phenotype.

A study of the chromosomal mapping of the human QIN gene, which was renamed by the Human Genome Organization Nomenclature Committee as FKH2, revealed that the QIN gene mapped to chromosome region 14q32. Fluorescence in situ hybridization techniques with a human QIN genomic clone refined the localization of human QIN gene to 14q13.

Synthetic peptides are used to detect a site on the mouse mammary tumour virus (MMTV)-1 superantigen that binds to class II major histocompatibility complex (MHC) antigens, which is surrounded by amino-acid residues 76-119 of the MMTV-1 superantigens. Experiments involving direct binding and competition reveal that the MMTV superantigen and Staphylococcal enterotoxin A (SEA) bind to atleast one common region on class II-MHC antigens.

A SPECIAL PLEASURE OF DOING SCIENCE COMES WHEN long-standing problems in seemingly unrelated areas flow together into a single problem. The association between certain retrovirues--the mammary tumor viruses--and superantigens is [...]

{/(CH3NH2)2Pt(1-MMeC)(9-Me-GH)~CL2-(1-MeC)}-0.5{/(1-MeCH)-(1-MeC)~Cl}-4.5H20, platinated DNA triplex, has been prepared by cocrystallization of trans-/(CH3NH2)2Pt(1-MeC-italN3)(9-MeGH-italN7)~Cl2 with 1-MeC. The triplex differs from tRNA tertiary base pairs. X-ray studies suggest the feasibility ofcovalent binding of an oligopyrimidine to a DNA duplex through a linear trans-(NH3)2Pt(II) entity. This may allow specific binding of the platinated oligonucleotide to DNA. Such possibility offers a solution to the problem of persistent fixation of the oligonucleotide to the target sequence, which is critical in DNA/RNA-based treatment of viral diseases.

The atomic force microscopy was used to characterize the basic features of mouse mammary tumor virus (MMTV) promoter nucleosomal arrays reconstituted with highly acetylated histones prepared from butyrate-treated HeLa cells. MMTV arrays containing highly acetylated histones show diminished intramolecular compaction compared to hypoacetylated MMTV arrays and no tendency for cooperativity in nucleosome occupation.