The oncoprotein P75gag-v-erbA represses thyroid hormone induced transcription only via response elements containing palindromic half-sites.

The v-erbA oncoprotein P75gag-v-erbA, derived from the thyroid hormone receptor alpha (TR alpha), functions as a transdominant transcriptional repressor. The mechanism by which P75gag-v-erbA acts is however poorly characterized. Here, we show that repression of TR alpha mediated transcription by P75gag-v-erbA in transformed erythroblasts is dependent on the structure of the thyroid hormone response element to which it binds. A very efficient repression was seen with hormone response elements having half-sites organized as everted repeats (ER), whereas repression was inefficient with directly repeated half-sites (DR). Promoters containing half-sites organized as an inverted palindrome (IR) gave an intermediate repression. Although P75gag-v-erbA failed to associate with the ligand binding domain of retinoid X (RXR) receptor in a two-hybrid test, the oncoprotein in nuclear extracts from transformed cells heterodimerised quantitatively with RXR upon binding to response elements of the DR type. On the other hand, both RXR/P75gag-v-erb heterodimers and other types of dimers formed on ER elements. P75gag-v-erbA also failed to bind to elements that contained only one half-site in vivo and in vitro. The data demonstrate that P75gag-v-erbA represses gene expression efficiently as a dimer, and suggest that thyroid hormone responsive genes that may be targets for the action of the oncoprotein are repressed most efficiently if they contain elements of the ER type.

Expression of T:G mismatch-specific thymidine-DNA glycosylase and DNA methyl transferase genes during development and tumorigenesis.

In situ hybridization was used to characterize the expression pattern of the T:G mismatch-specific thymidine-DNA glycosylase (TDG) gene, encoding a DNA repair enzyme which corrects G:T mismatches that result from the hydrolytic deamination of 5-methyl cytosines. TDG transcripts were uniformly and ubiquitously expressed from 7.5-13.5 days post-coitum, but were then markedly enriched in specific tissues of the developing fetus. At 14.5 gestational days, TDG was strongly expressed in the developing nervous system, thymus, lung, liver, kidney and intestine. At later stages, high levels of expression were detected in the thymus, brain, nasal epithelium and within proliferating regions of the intestine, skin, kidney, teeth and bone. This pattern of expression strongly correlated with those of the methyl transferase (MTase) gene, coding for the enzyme which specifically methylates CpG dinucleotides, and the p53 tumour suppressor gene. However, TDG and MTase were differentially expressed during maturation of the male and female germline. We also report that tumors occuring in mice which overexpress MMTV-v-Ha-ras or MMTV-c-myc transgenes or mice heterozygous for p53 gene disruption, all show elevated TDG and MTase expression specific to the transformed tissue.

Zonation of glucokinase in rat liver changes during postnatal development.

In the liver many metabolic pathways are preferentially localized in different zones of the acinus. It is assumed that this zonation allows an efficient adaptation to different states of nutrition, because alternative pathways can be regulated independently. It is reported that the rate limiting enzyme for the glycolytic pathway, glucokinase (EC 2.7.1.2), is predominantly located in the pericentral zone. The gene expression of glucokinase is induced to a maximum level after a carbohydrate-rich diet. In starved or diabetic rats glucokinase gene expression is barely detectable. In postnatal development glucokinase is induced to significant levels only from day 14 onwards. The distribution of the glucokinase protein in the rat liver lobule in the first 4 weeks of postnatal life was investigated by immunohistochemistry and compared to the distribution observed in adult rats. In adult rats considerably high levels of glucokinase are measureable as shown by immunoblotting utilizing a monospecific antibody and a photometric assay of glucokinase enzyme activity, respectively. Immunohistochemically the hepatic glucokinase protein is detected in the perivenous area. During postnatal development, the quantities of hepatic glucokinase protein and glucokinase enzyme activity start to increase significantly from day 15 onwards. Subsequently, glucokinase levels rise further until day 29. In contrast to the results obtained by immunoblotting, glucokinase is already detectable in some liver cells in sections from 6-day-old rats by immunohistochemistry. The liver lobule structure at this age is not completely developed, therefore it is not possible to definitely assign these cells to periportal or pericentral areas. At day 10 post partum the number of glucokinase expressing cells, which appear to be localized preferentially in the periportal zone, increases. In agreement with the immunoblotting, an immense increase in glucokinase activity was observed at day 14. The periportal zonation, clearly detectable at this time, remains stable until day 24. In sections from 29-day-old rats the periportal zonation begins to change into a more homogeneous pattern with a slight preference for periportal areas. The observed appearance of the periportal zonation of glucokinase during neonatal development is obviously in contrast to the perivenous expression of glucokinase in adult rats.

Identification of DNA binding sites for the V-erbA oncoprotein, the viral homolog to thyroid hormone receptor alpha.

The v-erbA oncogene protein, p75(gag-v-erbA), is a mutant form of the thyroid hormone receptor alpha (TR alpha) which has sustained mutations both in the ligand binding and DNA binding domains. The oncoprotein has therefore lost its ability to bind ligand, and its heterodimerization with the retinoid-X receptor (RXR) is impaired. Here, we have investigated the effects of the mutations in the DNA binding domain. By applying a PCR-based screening assay we isolated DNA sequences to which p75(gag-v-erbA) binds as a heterodimer with RXR, and characterized these with regard to their nucleotide sequence and ability to associate with RXR/P75(gag-v-erbA) heterodimers in vitro and in vivo. In the PCR selection assay the heterodimer exhibited a preference for direct repeats with a 3' half-site sequence AGGTCG and spacers of four or five nucleotides separating the two half-sites. These DNA binding data were confirmed by gel retardation assays with synthetic oligonucleotides as well as by transfection experiments using dominantly active VP16 fusion proteins with P75(gag-v-erbA) and TR alpha. The comparison between RXR/P75(gag-v-erbA) and RXR/TR alpha heterodimers demonstrated that although their DNA binding properties are very similar, however, a relaxed specificity of P75(gag-v-erbA) for the spacer length may allow it to interfere with more hormone signalling pathways than only that of thyroid hormone.

Normal hospital and low-bacterial diet in patients with cytopenia after intensive chemotherapy for hematological malignancy: a study of safety.

BACKGROUND: The purpose of this randomized, controlled pilot study is to address the question whether normal hospital diet (NHD) is safe when compared with low-bacterial diet (LBD) given to prevent infections in cytopenic patients who receive antimicrobial prophylaxis (AP). PATIENTS AND METHODS: The patients were randomized into two groups: one group to receive AP and LBD, the other to receive the same AP and NHD. The primary outcome parameter is colonization of the digestive tract with aerobic gram-negative bacilli and yeasts. Secondary outcome parameters were infections and total societal costs. RESULTS: No statistically significant differences between treatment groups were observed regarding the primary outcome parameter, gut colonization by yeasts or gram-negative bacilli, or infections, use of antimicrobials, days with fever and total societal costs. CONCLUSION: On the basis of the results of this pilot study, NHD appears to be as safe as LBD in patients with chemotherapy-induced cytopenia. Furthermore, the results indicate that LBD may offer no additional benefit as an infection preventive measure to the measures already implemented, such as AP. Thus, a larger randomized study, powered adequately to determine noninferiority of NHD to LBD is warranted and safe to be carried out.

Home care versus hospital care of patients with hematological malignancies and chemotherapy-induced cytopenia.

BACKGROUND: The purpose of this review study is to examine the accumulating evidence of safety of home care, with regard to infection-related morbidity and mortality, for patients with chemotherapy-induced cytopenia, in light of previous studies on the necessity of protective isolation (PI). PATIENTS AND METHODS: The existing literature on PI, and home care of cytopenic patients after chemotherapy, published in the English language, based on a Medline search, is reviewed. RESULTS: The studies published so far on home care versus hospital care are all non-randomized studies and confirm that home care of cytopenic patients is safe, in terms of morbidity and mortality due to infections. On the other hand, the majority of studies on the comparison of PI with standard hospital care conclude that an infection-preventive effect of PI exists. The pooled statistics performed confirmed that such an effect of PI exists regarding the occurrence of severe infections, although no benefit to mortality has been shown. CONCLUSIONS: Regarding home care, only the results of a prospective, randomized study of sufficient power will enable definitive conclusions to be drawn as to whether home care is equally safe as hospital-based care with PI.

Suppression of c-fos precursor RNA splicing by the protein kinase C inhibitor H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine].

In JB6 epidermal cells, induction of fos proto-oncogene expression by phorbol 12-myristate 13-acetate can be inhibited by the protein kinase C (PKC) inhibitor H7 [1-5(isoquinolinesulphonyl)-2-methylpiperazine]. The compound causes also a dose-dependent suppression of fos precursor RNA splicing which, however, appears to react somewhat less sensitively to H7 than does PKC activity. This indicates that H7-induced accumulation of fos precursor RNA is not due to inhibition of PKC. Support for this interpretation comes from the finding that other inhibitors of PKC, such as N-(2-guanidinoethyl)-5-isoquinolinesulphonamide dihydrochloride, sphingosine, staurosporine or N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide, do not suppress splicing when applied at PKC-inhibiting concentrations.

Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.

A better method was developed for analysis and identification of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins, bound to nitrocellulose, were identified by immunoblotting, while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography. The technique readily identified thyroid hormone receptors interacting with response elements representing inverted or direct repeats of the consensus half-site AGGTCA. In addition, specific antisera identified both the thyroid hormone and the retinoic acid receptors in heterodimeric complexes. Adding a third membrane and digoxigenin-labeled DNA probes allowed separate detection of [125I]T3 (labeled 3,5,3'-L-triiodothyronine), DNA, and protein from a single gel-shift reaction. The usefulness of this technique was also demonstrated by detecting the transcription factors P75gag-v-erbA and Jun in shifted complexes. Finally, proteins and DNA transferred to anion-exchange membranes can be eluted and subjected to further study. The combination of the gel shift and the immunoblot approaches (called "Shift-Western blotting") allows identification of the individual components of protein-DNA complexes containing multiple transcription factors, their cognate DNA elements and, when applicable, also the ligand.

Intracellular localization of the Ret finger protein depends on a functional nuclear export signal and protein kinase C activation.

The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Calpha (PKCalpha) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.

Transactivation by the thyroid hormone receptor is dependent on the spacer sequence in hormone response elements containing directly repeated half-sites.

The thyroid hormone receptor (TR) regulates the transcription of its target genes by interacting with specific hormone response elements consisting usually of directly repeated half-sites with the consensus sequence AGGTCA. To investigate the role of the spacer sequences separating the half-sites, heterodimers formed by TRalpha and the retinoid-X receptor (RXR) were used in a PCR based selection and amplification assay. The TRalpha/RXR heterodimer selected for elements with directly repeated half-sites having a spacer of 4 nucleotides (DR4). Preferences for nucleotides in the TR binding half-site motif as well as for the 4 nucleotides separating the two half-sites were found. DNA binding and transfection studies using DR4 elements with different spacer sequences showed the importance of these nucleotides for the activity of the response element: some spacer sequences allowed little or no transactivation from the element, whereas other sequences supported strong transactivation. A pyrimidine nucleotide in position three of the spacer enhanced TRalpha binding and transactivation. Additional experiments showed that heterodimers between RXR and other putative receptors exhibited a similar but distinct specificity for the spacer sequence. Our results thus suggest that the four nucleotides separating the two half-sites in hormone response elements have a major role in determining induction of hormone responsive genes.

Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta.

The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.

The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors.

Nuclear receptors can function as ligand-inducible transregulators in both mammalian and yeast cells, indicating that important features of control of transcription have been conserved throughout evolution. Here, we report the isolation and characterization of a yeast protein that exhibits properties expected for a coactivator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of the retinoid X (RXRalpha) and estrogen (ERalpha) receptors. This protein is identical to Ada3, a component of the yeast Ada coactivator complex. We demonstrate that: (1) the region encompassing residues 347-702 of Ada3 interacts with the LBD of RXRalpha and ERalpha in a ligand-dependent manner in yeast; (2) this interaction corresponds to a direct binding and requires the integrity of the core of the AF-2 activating domain (AF-2 AD) of both RXRalpha and ERalpha; (3) Ada3 as well as Ada2 and Gcn5, two other components of the Ada complex, are required for maximal AF-2 activity in yeast; and (4) Ada3 is able to enhance the AF-2 activity of RXRalpha and ERalpha when overexpressed in yeast and mammalian cells. Taken together, these data indicate that ligand-dependent transactivation by RXRalpha and ERalpha in yeast is mediated at least in part by the Ada complex, in which the Ada3 subunit directly binds to the holoreceptor LBD.

Tumor promotion and depletion of protein kinase C in epidermal JB6 cells.

Promotion of JB6 epidermal cells to anchorage-independent growth requires exposure to TPA for greater than 4 days. Over a similar time span, a practically complete loss of enzymic and immunoreactive proteinkinase C (PKC) equivalents was observed at greater than 10 nM TPA. Promotion did not appear to require (transient) activation of PKC since PKC inhibitors H7 and HA1004 did not prevent but enhanced colony formation in soft agar at concentrations greater than IC50-values. The efficacy of the inhibitors in vivo was shown by their ability to suppress PKC-induced transcription of c-fos gen. PKC inhibitors that interfered with cell proliferation at lower concentrations than those required for PKC inhibition (sphingosine, staurosporin, sangivamycin, trifluoperazine) did not stimulate anchorage-independent growth. As H7 as well as HA1004 were able to promote JB6 cells in the complete absence of TPA, and induced neither depletion nor processing of PKC we postulate that depletion/inactivation rather than activation of PKC correlates with the promotion of epidermal JB6 cells to anchorage-independent growth.

Retinoic acid receptors interact physically and functionally with the T:G mismatch-specific thymine-DNA glycosylase.

The pleiotropic effects of retinoids are mediated by nuclear receptors that are activated by 9-cis- or all-trans-retinoic acid to function as ligand-dependent transcription factors. In a yeast one-hybrid screen for proteins capable of interacting with native retinoic acid receptor (RAR), we have isolated the T:G mismatch-specific thymine-DNA glycosylase (TDG), which initiates the repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. Here, we report that TDG can interact with RAR and the retinoid X receptor (RXR) in a ligand-independent manner, both in yeast and in vitro. Mapping of the binding sites revealed interaction with a region of the ligand binding domain harboring alpha-helix 1 in both RAR and RXR. In transient transfection experiments, TDG potentiated transactivation by RXR from a direct repeat element spaced by one nucleotide (DR1) and by RXR/RAR heterodimers from a direct repeat element spaced by five nucleotides (DR5). In vitro, TDG enhanced RXR and RXR/RAR binding to their response elements. These data indicate that TDG is not only a repair enzyme, but could also function in the control of transcription.

Epigenetic activation of Gi-2 protein, the product of a putative protooncogene, mediates tumor promotion in vitro.

Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.