RINF (CXXC5) is overexpressed in solid tumors and is an unfavorable prognostic factor in breast cancer.

BACKGROUND: We have previously described the essential role of the retinoid-inducible nuclear factor (RINF) during differentiation of hematopoietic cells and suggested its putative involvement in myeloid leukemia and preleukemia. Here, we have investigated whether this gene could have a deregulated expression in malignant tissues compared with their normal tissues of origin and if this potential deregulation could be associated with important clinicopathological parameters. PATIENTS AND METHODS: RINF messenger RNA expression was examined in biopsies from locally advanced breast tumors, metastatic malignant melanomas, and papillary thyroid carcinomas and compared with their paired or nonpaired normal reference samples. Further, the prognostic role of RINF expression was evaluated in locally advanced breast cancer. RESULTS: RINF expression was significantly higher in all tumor forms (primary breast, and thyroid cancers and metastatic melanomas) as compared with normal control tissues (P < 0.001 for each comparison). Importantly, high levels of RINF expression correlated to a poor overall survival in breast cancer (P = 0.013). This finding was confirmed in three independent public microarray datasets (P = 0.043, n = 234; P = 0.016, n = 69; P = 0.001, n = 196) and was independent of tamoxifen therapy. Notably, high levels of RINF was strongly associated with TP53 wild-type status (P = 0.002) possibly indicating that high levels of RINF could substitute for TP53 mutations as an oncogenic mechanism during the malignant development of some cases of breast cancer. CONCLUSIONS: Our data indicate that (i) RINF overexpression is associated with the malignant phenotype in solid tumors and (ii) RINF overexpression represents an independent molecular marker for poor prognosis in breast tumors.

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper.

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-alpha-acetyltransferase complex.

Protein N-epsilon-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-epsilon-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-epsilon-acetyltransferases, the N-alpha-acetyltransferases transferring acetyl groups to the alpha-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH-hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH-hARD1 complex in cell survival and underscore the importance of protein N-alpha-acetylation in mammalian cells.

Disposition of endogenous homocysteine by mouse fibroblast C3H/10T1/2 Cl 8 and the chemically transformed C3H/10T1/2 MCA Cl 16 cells following methotrexate exposure.

The tumorigenic cell line termed "MCA Cl 16" was derived from C3H/10T1/2 clone (Cl) 8 cells by chemical transformation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (Cl 16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counterpart Cl 8 cells. The disposition of endogenous L-homocysteine (Hcy) was investigated in these two cell lines after MTX exposure. Both nonmalignant and transformed cells exported Hcy into the extracellular medium, and only small amounts were retained within the cells. The Hcy efflux from the malignant cells was markedly increased after MTX exposure (0.5-10 microM), and this effect was almost completely prevented by 5-formyl-tetrahydrofolate (THF), whereas treatment with thymidine plus hypoxanthine did not inhibit the MTX-dependent Hcy efflux. Cytotoxic concentration of MCA reduced rather than increased the Hcy efflux from these cells. High concentrations of MTX (greater than 10 microM) were required to increase the release of Hcy from nonmalignant cells. The enhancement of Hcy export from the malignant cells in the presence of MTX was not associated with cellular build-up of S-adenosyl-L-homocysteine (AdoHcy), indicating that the amount of intracellular Hcy was kept below the level required for inhibition or reversion of the AdoHcy hydrolase reaction. MTX-dependent Hcy efflux probably reflects cellular deficiency of 5-methyl-THF required for the salvage of Hcy to methionine and may therefore be a measure of lack of this reduced folate relative to the metabolic demand.

Expression of c-erbB-3 and c-erbB-4 proteins in papillary thyroid carcinomas.

c-erbB-3 and c-erbB-4 protein expression was analyzed using immunohistochemistry in 138 fresh-frozen thyroid tissue samples from 106 patients, including 56 cases of papillary thyroid carcinoma. Increased expression of c-erbB-3 and c-erbB-4 proteins was observed in papillary carcinomas compared to nonneoplastic thyroid tissue. No amplifications of the c-erbB-3 and c-erbB-4 genes were detected. Coexisting overexpression of epidermal growth factor receptor, c-erbB-2, c-erbB-3, and c-erbB-4 was demonstrated in 36 (64%) of 56 papillary thyroid carcinomas. These findings suggest a common regulatory mechanism for the type I (epidermal growth factor receptor-related) receptors in papillary thyroid carcinomas and provide numerous possibilities for functional receptor interactions.

Enhanced expression of c-myc and decreased expression of c-fos protooncogenes in chemically and radiation-transformed C3H/10T1/2 Cl 8 mouse embryo cell lines.

c-abl, c-fos, c-Ha-ras, c-myc, and c-mos were expressed whereas c-sis, c-fms, c-rel, c-src, and c-myb expression was not detectable in C3H/10T1/2 Cl 8 (10T1/2) cells and in eight chemically and radiation-transformed 10T1/2 cell lines. The expression of c-abl, c-fos, c-Ha-ras, and c-myc was growth-related in nontransformed 10T1/2 cells. c-abl and c-fos expression increased at confluence by 5- and 9-fold, respectively, compared to that in log phase cells. c-Ha-ras and c-myc transcripts were most abundant in log phase cells and decreased by 70 and 50%, respectively, in confluent cells. There were no significant growth-related changes in the expression of c-Ha-ras, c-myc, or c-abl in methylcholanthrene-transformed Cl 15 cells. The c-fos transcript was not detected in Cl 15 cell cultures. c-abl, c-fos, c-ras, and c-myc were expressed in whole C3H mouse embryo tissue, mouse liver, and 10T1/2 cells. Sizes of these protooncogene transcripts in 10T1/2 cells were the same as those in whole embryo tissue, except that 10T1/2 cells did not express the 8.2-kilobase abl transcript. At subconfluence, equivalent low levels of c-mos expression were observed in nontransformed and in the eight transformed 10T1/2 cell lines. The level of c-abl expression was similar in the nontransformed and in the eight transformed cell lines, but there was a new 8.2-kilobase transcript in the transformed MCA Cl 15 cell line. c-fos was expressed in 10T1/2 cells but was not detectable or greatly reduced in eight transformed cell lines. c-Ha-ras was expressed to a similar extent in eight transformed cell lines and in nontransformed 10T1/2 cells. In the UVC-4 transformed cell line, extra 3.3-kilobase Ha-ras and 7.5-kilobase Ki-ras transcripts were observed. c-myc was expressed at 4- to 7-fold higher levels in six transformed cell lines compared to 10T1/2 cells. There were no major rearrangements in or amplification of the c-myc gene in three transformed cells overexpressing this gene 5-fold. These studies show that enhanced expression of c-myc and decreased expression of c-fos correlate with the chemically and radiation transformed states of 10T1/2 cells. Changes in c-fos and c-myc oncogene expression may be casually linked to late stages of neoplastic transformation in these chemically and radiation transformed 10T1/2 cell lines.

Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer.

TP53 status [mutations, immunostaining, and loss of heterozygosity (LOH)], expression of c-erbB-2, bcl-2, and histological grading were correlated to the response to doxorubicin monotherapy (14 mg/m2) administered weekly to 90 patients with locally advanced breast cancer. Mutations in the TP53 gene, in particular those affecting or disrupting the loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (P = 0.063 for all mutations and P = 0.008 for mutations affecting L2/L3, respectively). Similarly, expression of c-erbB-2 (P = 0.041), a high histological grade (P = 0.023), and lack of expression of bcl-2 (P = 0.018) all predicted chemoresistance. No statistically significant association between either p53 immunostaining or TP53 LOH and response to therapy was recorded, despite the finding that both were associated with TP53 mutation status (p53 immunostaining, P < 0.001; LOH, P = 0.021). Lack of immunostaining for p53 despite mutation of the TP53 gene was particularly seen in tumors harboring nonsense mutations or deletions/splices (7 of 10 negative for staining compared with 4 of 16 with missense mutations). TP53 mutations (total/affecting L2/L3 domains) were associated with expression of c-erbB-2 (P < 0.001 for both), high histological grade (P = 0.001 and P = 0.025), and bcl-2 negativity (P = 0.003 and P = 0.002). TP53 mutations, histological grade, and expression of bcl-2 (but not LOH or c-erbB-2 expression) all predicted for relapse-free as well as breast cancer-specific survival in univariate analysis (Ps between <0.0001 and 0.0155), but only tumor grade was found to be predictive in multivariate analysis (P = 0.01 and P = 0.0007, respectively). Our data are consistent with the hypothesis that certain TP53 mutations predict for resistance to doxorubicin in breast cancer patients. However, the observation that the majority of patients with TP53 mutations affecting or disrupting the L2/L3 domains with LOH in addition (n = 12) obtained a partial response (n = 4) or stabilization of disease (n = 5) during chemotherapy suggests redundant mechanisms to compensate for loss of p53 function. Our findings are consistent with the hypothesis that other defects may act in concert with loss of p53 function, causing resistance to doxorubicin in breast cancers.

Differential changes of retinoid-X-receptor (RXR alpha) and its RAR alpha and PML-RAR alpha partners induced by retinoic acid and cAMP distinguish maturation sensitive and resistant t(15;17) promyelocytic leukemia NB4 cells.

The expression of retinoid receptors (RXRalpha, RARalpha and the chimeric form PML-RARalpha) was analysed both at the mRNA and protein level in the maturation sensitive NB4 and resistant NB4-R1 cell lines of t(15;17) promyelocytic leukemia (APL). All-trans RA and cAMP which show synergistic activity in inducing maturation of NB4 cells and maturation triggering of the RA 'primed' NB4-R1 resistant cells, distinctly modulate RXRalpha, RARalpha and PML-RARalpha mRNA. In the NB4 and NB4-R1 cells, RXRalpha mRNA was downregulated by RA, but only in RA-primed NB4-R1 cells a release from RXRalpha mRNA downregulation was obtained by cAMP treatment. RXRalpha protein (53 kDa) was decreased to the western-blot detection limit (97.5%) by RA in NB4 cells, but in NB4-R1 cells although it was frankly decreased (85%), the signal for RXRalpha protein remained very significant. More importantly, while cAMP slightly upregulated RXRalpha protein in RA-treated NB4 cells, it caused an increase of RXRalpha protein in RA-treated NB4-R1 cells bringing RXRalpha to the initial control level. RXRalpha partners in heterodimers (PML-RARalpha, RARalpha) were also analysed. In contrast to RXRalpha, RARalpha and PML-RARalpha mRNA were not modulated by RA and/or cAMP, while significant changes were observed at the protein levels. A putatively phosphorylated form of RARalpha (52 kDa) decreased during maturation of NB4 cells, but was unchanged in resistant NB4-R1 cells. Conversely, while PML-RARalpha remained stable during RA-induced NB4 maturation, RA treatment which failed to induce maturation of NB4-R1 cells significantly down-regulated the chimeric receptor (120 kDa). These differences most likely results from translational and post-translational regulation. This work reveals complex pattern of subtle changes at the protein level distinguishing RA-sensitive and RA-resistant cells. Our data show that the RA-cAMP synergistic effect on NB4 cell maturation and cooperation in triggering maturation of RA-primed NB4-R1 cells operate changes in the RXR/PML-RARalpha ratio which are both favouring RXRalpha. In both cell lines, variations of PML-RARalpha and RXRalpha may result in a decrease in the formation of the PML-RARalpha/RXRalpha heterodimers which are supposed involved in the block of maturation. This may prove crucial to embark cells on maturation.

cAMP signalling is decisive for recovery of nuclear bodies (PODs) during maturation of RA-resistant t(15;17) promyelocytic leukemia NB4 cells expressing PML-RAR alpha.

We analysed the expression of retinoid receptors and PML in relation to the morphology of PML-containing nuclear bodies (PODs) in maturation sensitive (NB4) and resistant subclones (NB4-R1 and R2) of the promyelocytic NB4 cell line. The basal level of RARalpha, RXRalpha and PML mRNA and protein were roughly the same in the three cell lines. While NB4 and NB4-R1 cells express comparable amounts of PM-RARalpha mRNA and 120 kDa protein, NB4-R2 cells despite normal mRNA levels the 120 kDa protein was not detectable. In NB4-R2 cells however, two novel PML-related entities of 65 kDa and 85 kDA were detected with a anti-PML antibody, in addition to the two PML isoforms of 78 and 97 kDa found in any NB4 cells. Despite the 120 kDa PML-RARalpha defect, NB4-R2 cells show micropunctuated nuclear bodies typical of APL cells. Contrasting with NB4 cells, neither NB4-R1 cells which express PML-RARalpha, nor NB4-R2 cells lacking the 120 kDa PML-RARalpha reorganised nuclear bodies (PODs) in response to RA. Importantly, in RA-primed NB4-R1 cells, a secondary event triggered by cAMP restored PODs, concomitant to maturation. This indicates that the recovery of nuclear bodies in APL is dissociated from the early action of RA in cell maturation. Finally, the key finding of this work is that cAMP signalling ultimately determines the recovery of nuclear bodies associated to cell maturation.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT-PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cysteine-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.

Inhibition of T4 polynucleotide kinase by the ATP analog, beta, gamma-imidoadenylyl 5'-triphosphate.

The inhibition of T4 polynucleotide kinase by beta,gamma-imidoadenylyl 5'-triphosphate has been investigated. It was found that the ATP analog was a competitive inhibitor with regard to ATP and a noncompetitive inhibitor with regard to DNA possessing a 5'-hydroxyl group. At pH 8.0, the Ki values were 3 and 11 mM, respectively. beta,gamma-imidoadenylyl 5'-triphosphate was not a substrate in the forward reaction, but would replace ADP and ATP in the reverse reaction. The reverse reaction was also used to make beta,gamma-imidoadenylyl 5'-tetraphosphate.

Purification and kinetic properties of polynucleotide kinase from rat testes.

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.

Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.

Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis.

The C3H/10T1/2 Cl8 HAbetaC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAbetaC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of alpha/betaPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAbetaC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAbetaC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [(14)C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between alpha/betaPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

A mutated PML/RARA found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks RARA and wild-type PML/RARA transcriptional activities.

The fusion protein PML/RARA, associated with acute promyelocytic leukemia behaves as an abnormal retinoic acid (RA) receptor with altered transactivation properties but is still inducible by RA. The chimeric protein is thought to promote leukemogenesis but also paradoxically to mediate the sensitivity to ATRA of APL cells. This has been supported by works reporting that in vitro ATRA resistance is characterized by defects in the RARA/E-domain of PML/RARA. In the present report, we identified a new mutation in the E domain of PML/RARA which is associated with a RA-resistant subline of NB4 cells; NB4-R2. This mutation, identical to the Gln411 mutation found in HL60-R, changes the amino acid Gln903 to an in-phase stop codon, generating a truncated form of PML/RARA which has lost 52 amino acids at its C-terminal end. We have studied the effect of the truncated PML/RARA protein on PML NB formation and RARA and PML/RARA transcriptional activity. We show here that the fusion mutant exerts a dominant negative effect on wild-type PML, PML/RARA and RARA transcription activity. These findings highlight the important role of the RARA E-domain of PML/RARA in mediating RA sensitivity in APL cells.

The nuclear-associated endoplasmic reticulum is an early target for the action of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in C3H/10T1/2 fibroblasts.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) had a dual effect on the cellular membranes of C3H/10T1/2 cells in that it caused both a stimulation of [3H]choline incorporation and an enhancement of the solubilization of choline from prelabelled cells. Subfractionation studies showed that the release of [3H]choline occurred almost exclusively from nuclear-associated endoplasmic reticulum. The release was dependent on the presence of Mg2+ and Ca2+, indicating an enzyme-mediated reaction. In vivo, TPA stimulated the incorporation of [3H]choline into all subcellular fractions. The data indicate that the nuclear-associated endoplasmic reticulum represents an early target for TPA action.

Absence of abnormalities of the c-erbB-1 and c-erbB-2 proto-oncogenes in human thyroid neoplasia.

The c-erbB-1 and c-erbB-2 proto-oncogenes are frequently activated by gene amplification and overexpression in a variety of human cancers. In an analysis of a large series of benign and malignant thyroid tumours, no abnormalities of structure or expression of either of c-erbB-1 or c-erbB-2 were found. Activation of these oncogenes is not a necessary event in neoplasia of this epithelial system.

Evidence against involvement of APC mutation in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is one of several tumours associated with familial adenomatous polyposis (FAP), an inherited tumour syndrome which appears to result from germ-line mutation of the APC tumour suppressor gene. Here we investigate the possibility that somatic mutation of APC might play a role in sporadic PTC. 16 cases of PTC together with matched normal tissue were examined by single-strand conformation polymorphism (SSCP) analysis, concentrating on the mutation cluster region (MCR) of the APC gene (codons 1286-1513). No evidence of mutation was observed in any sample. We conclude that APC mutation, at least in the MCR, is not a significant causal mechanism in sporadic PTC.

Early modulation of genes encoding peroxisomal and mitochondrial beta-oxidation enzymes by 3-thia fatty acids.

The aim of the present study was to elucidate the effects of a single dose of 3-thia fatty acids (tetradecylthioacetic acid and 3-thiadicarboxylic acid) over a 24-hr study period on the expression of genes related to peroxisomal and mitochondrial beta-oxidation in liver of rats. The plasma triglyceride level decreased at 2-4 hr, 4-8 hr, and 8-24 hr, respectively, after a single dose of 150, 300, or 500 mg of 3-thia fatty acids/kg body weight. Four to eight hours after administration of 3-thia fatty acids, a several-fold-induced gene expression of peroxisomal multifunctional protein, fatty acyl-CoA oxidase (EC 1.3.3.6), fatty acid binding protein, and 2,4-dienoyl-CoA reductase (EC 1.3.1.43) resulted, concomitant with increased activity of 2,4-dienoyl-CoA reductase and fatty acyl-CoA oxidase. The expression of carnitine palmitoyltransferase-I and carnitine palmitoyltransferase-II increased at 2 and 4 hr, respectively, although at a smaller scale. In cultured hepatocytes, 3-thia fatty acids stimulated fatty acid oxidation after 4 hr, and this was both L-carnitine- and L-aminocarnitine-sensitive. The hepatic content of eicosapentaenoic acid and docosahexaenoic acid decreased throughout the study period. In contrast, the hepatic content of oleic acid tended to increase after 24 hr and was significantly increased after repeated administration of 3-thia fatty acids. Similarly, the expression of delta9-desaturase was unchanged during the 24-hr study, but increased after feeding for 5 days. To conclude, carnitine palmitoyltransferase-I expression seemed to be induced earlier than 2,4-dienoyl-CoA reductase and fatty acid binding protein, and not later than the peroxisomal fatty acyl-CoA oxidase. The expression of delta9-desaturase showed a more delayed response.