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Biomedical subjects

I Hoffmann

Publications and source records attributed to I Hoffmann.

At least 19 recordsLinked to original sources

Molecular analysis of two closely related mouse aldehyde dehydrogenase genes: identification of a role for Aldh1, but not Aldh-pb, in the biosynthesis of retinoic acid.

Mammalian class I aldehyde dehydrogenase (ALDH1) has been implicated as a retinal dehydrogenase in the biosynthesis of retinoic acid, a modulator of gene expression and cell differentiation. As the first step towards studying the regulation of ALDH1 and its physiological role in the biosynthesis of retinoic acid, mouse ALDH1 cDNA and genomic clones have been characterized. During the cloning process, an additional closely related gene was also isolated and named Aldh-pb, owing to its high amino acid sequence identity (92%) with the rat phenobarbitol-inducible ALDH protein (ALDH-PB). Aldh1 spans about 45 kb in length, whereas Aldh-pb spans about 35 kb. Both genes are composed of 13 exons, and the positions of all the exon/intron boundaries are conserved with those of human ALDH1. The promoter regions of Aldh1 and Aldh-pb demonstrate high sequence similarity with those of human ALDH1 and rat ALDH-PB. Expression of Aldh1 and Aldh-pb is tissue-specific, with mRNAs for both genes being found in the liver, lung and testis, but not in the heart, spleen or muscle. Expression of Aldh-pb, but not Aldh1, was also detected at high levels in the kidney. Aldh1 and Aldh-pb encode proteins of 501 amino acids with 90% positional identity. To examine the relative roles of these two enzymes in retinoic acid synthesis in vivo, Xenopus embryos were injected with mRNAs encoding these enzymes to assay the effect on conversion of endogenous retinal into retinoic acid. Injection of ALDH1, but not ALDH-PB, mRNA stimulated retinoic acid synthesis in Xenopus embryos at the blastula stage. Thus our results indicate that Aldh1 can function in retinoic acid synthesis under physiological conditions, but that the closely related Aldh-pb does not share this property.

Aldehyde Dehydrogenase

Immunohistological analysis of E-cadherin, alpha-, beta- and gamma-catenin expression in colorectal cancer: implications for cell adhesion and signaling.

Intercellular adhesion mediated by the E-cadherin/catenin complex is a prerequisite for epithelial integrity and differentiation. In carcinomas, E-cadherin function is frequently disturbed, and has been suggested to increase invasion and metastasis of tumour cells. beta-catenin has also been implicated in signaling pathways essential for tumour formation. We analysed the E-cadherin/catenin adhesion system of colorectal tumours at different clinical stages. In primary carcinomas (n = 91), there was a frequent reduction in E-cadherin (44%) and alpha-catenin expression (36%). In contrast, beta-catenin and gamma-catenin expression were seldom reduced (4% and 15%, respectively). Similar expression patterns were observed in liver metastases from unrelated colorectal tumours (n = 27). There was a significant relationship between loss of E-cadherin and alpha-catenin expression and poorly differentiated (G3-4) tumours. Our results suggest that reduction of E-cadherin/alpha-catenin expression is a frequent event in primary and metastatic colorectal carcinomas. Furthermore, beta-catenin expression remains normal in colorectal cancer, suggesting the essential role of beta-catenin in signaling pathways.

Cadherins

Alcohol dehydrogenases in Xenopus development: conserved expression of ADH1 and ADH4 in epithelial retinoid target tissues.

Mammalian alcohol dehydrogenases ADH1 (class I ADH) and ADH4 (class IV ADH) function as retinol dehydrogenases contributing to the synthesis of retinoic acid, the active form of vitamin A involved in growth and development. Xenopus laevis ADH1 and ADH4 genes were isolated using polymerase chain reaction primers corresponding to conserved motifs of vertebrate ADHs. The predicted amino acid sequence of Xenopus ADH1 was clearly found to be an ortholog of ADH1 from the related amphibian Rana perezi. Phylogenetic tree analysis of the Xenopus ADH4 sequence suggested this enzyme is likely to be an ADH4 ortholog, and this classification was more confidently made when based also on the unique expression patterns of Xenopus ADH1 and ADH4 in several retinoid-responsive epithelial tissues. Northern blot analysis of Xenopus adult tissues indicated nonoverlapping patterns of ADH expression, with ADH1 mRNA found in small intestine, large intestine, liver, and mesonephros and ADH4 mRNA found in esophagus, stomach, and skin. These nonoverlapping tissue-specific patterns are identical to those previously observed for mouse ADH1 and ADH4, thus providing further evidence that Xenopus ADH1 and ADH4 are orthologs of mouse ADH1 and ADH4, respectively. During Xenopus embryonic development ADH1 mRNA was first detectable by Northern blot analysis at stage 35, whereas ADH4 mRNA was undetectable through stage 47. Whole-mount in situ hybridization indicated that ADH1 expression was first localized in the pronephros during Xenopus embryogenesis, thus conserved with mouse embryonic ADH1 which is first expressed in the mesonephros. ADH4 expression was not detected in Xenopus embryos by whole-mount in situ hybridization but was localized to the gastric mucosa of the adult stomach, a property shared by mouse ADH4. Conserved expression of ADH1 and ADH4 in retinoid-responsive epithelial tissues of amphibians and mammals argue that these enzymes may perform essential retinoid signaling functions during development of the pronephros, mesonephros, liver, and lower digestive tract in the case of ADH1 and in the skin and upper digestive tract in the case of ADH4.

Alcohol Dehydrogenase

The cdc25B phosphatase is essential for the G2/M phase transition in human cells.

Cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. In human cells, cdc25 proteins are encoded by a multigene family, consisting of cdc25A, cdc25B and cdc25C. While cdc25A plays a crucial role at the G1/S phase transition, cdc25C is involved in the dephosphorylation and activation of the mitotic kinase, cdc2/cyclinB. In addition, cdc25C itself is regulated by cdc2/cyclinB which then creates a positive feedback loop that controls entry into mitosis. In this study we show that the activity of cdc25B appears during late S phase and peaks during G2 phase. Both in vitro and in vivo cdc25B is activated through phosphorylation during S-phase. Using a cell duplication, microinjection assay we show that ablation of cdc25B function by specific antibodies blocks cell cycle progression in Hs68 cells by inhibition of entry into mitosis. Cdc25B function neither plays a role in later stages of mitosis nor for the inititation of DNA replication. These results indicate that cdc25B is a mitotic regulator that might act as a 'starter phosphatase' to initiate the positive feedback loop at the entry into M phase.

Antibodies

A beta-catenin mutation in a sporadic colorectal tumor of the RER phenotype and absence of beta-catenin germline mutations in FAP patients.

As a signaling protein in the Wnt pathway beta-catenin plays a crucial role in the regulation of cellular proliferation. Recently, oncogenic beta-catenin mutations were described in human colorectal cancer and melanoma cell lines. Since activating mutations in the beta-catenin gene have similar effects on the biochemical level as inactivating mutations in the tumor suppressor gene APC, it is speculated that beta-catenin mutations may substitute APC gene inactivation in carcinogenesis. To address this question we analyzed twenty-three sporadic colorectal tumors of different progression states for mutations in the beta-catenin gene. Eighteen of these tumors showed the wildtype APC gene sequence. In only one of the tumors with wildtype APC a beta-catenin gene mutation was found. This tumor was of the RER (replication error) phenotype which may explain the finding that the mutation occurred in a sequential repeat motif of the beta-catenin gene. The second aim of this study was to investigate whether differences in the phenotypic variability in FAP (familial adenomatous polyposis coli) might be due to inherited alterations in the beta-catenin gene. For this we analyzed DNA from fourteen FAP patients from eight different families for germline mutations in the beta-catenin gene. We did not find any beta-catenin gene alteration in these samples. Our results indicate that somatic beta-catenin activating mutations contribute only to a minor part of human colorectal tumors and that germline beta-catenin mutations do not play a role in the variability of symptoms in FAP.

Adenomatous Polyposis Coli

Detection of HHV-8 DNA in a German patient with classical Kaposi's sarcoma may allow an estimation of the incubation period.

DNA of HHV-8 (Kaposi's sarcoma-associated Herpes virus [KSHV]) was detected in a biopsy of a Kaposi's sarcoma in an elderly male patient from Saxony (East Germany). The diagnosis of classical Kaposi's sarcoma was first made in 1986. During World War II, the patient had been on active service on the Greek Islands of Crete and Rhodes only, he did not travel outside East Germany after the war. It is assumed that the patient was infected during his stay on the islands of Crete or Rhodes, where classical Kaposi's sarcoma is endemic. If so, the incubation period of classical Kaposi's sarcoma could be as long as 40 years.

Aged

The changing cigarette, 1950-1995.

Nicotine is recognized to be the major inducer of tobacco dependence. The smoking of cigarettes as an advantageous delivery system for nicotine, accelerates and aggravates cardiovascular disease, and is causally associated with increased risks for chronic obstructive lung disease, cancer of the lung and of the upper aerodigestive system, and cancer of the pancreas, renal pelvis, and urinary bladder. It is also associated with cancer of the liver, cancer of the uterine cervix, cancer of the nasal cavity, and myeloid leukemia. In 1950, the first large-scale epidemiological studies documented that cigarette smoking induces lung cancer and described a dose-response relationship between number of cigarettes smoked and the risk for developing lung cancer. In the following decades these observations were not only confirmed by several hundreds of prospective and case-control studies but the plausibility of this causal association was also supported by bioassays and by the identification of carcinogens in cigarette smoke. Whole smoke induces lung tumors in mice and tumors in the upper respiratory tract of hamsters. The particulate matter of the smoke elicits benign and malignant tumors on the skin of mice and rabbits, sarcoma in the connective tissue of rats, and carcinoma in the lungs of rats upon intratracheal instillation. More than 50 carcinogens have been identified, including the following classes of compounds: polynuclear aromatic hydrocarbons (PAH), aromatic amines, and N-nitrosamines. Among the latter, the tobacco-specific N-nitrosamines (TSNA) have been shown to be of special significance. Since 1950, the makeup of cigarettes and the composition of cigarette smoke have gradually changed. In the United States, the sales-weighted average "tar" and nicotine yields have declined from a high of 38 mg "tar" and 2.7 mg nicotine in 1954 to 12 mg and 0.95 mg in 1992, respectively. In the United Kingdom, the decline was from about 32 mg "tar" and 2.2 mg nicotine to less than 12 mg "tar" and 1.0 mg nicotine per cigarette. During the same time, other smoke constituents changed correspondingly. These reductions of smoke yields were primarily achieved by the introduction of filter tips, with and without perforation, selection of tobacco types and varieties, utilization of highly porous cigarette paper, and incorporation into the tobacco blend of reconstituted tobacco, opened and cut ribs, and "expanded tobacco." In most countries where tobacco blends with air-cured (burley) tobacco are used, the nitrate content of the cigarette tobacco increased. In the United States nitrate levels in cigarette tobacco rose from 0.3-0.5% to 0.6-1.35%, thereby enhancing the combustion of the tobacco. More complete combustion decreases the carcinogenic PAH, yet the increased generation of nitrogen oxides enhances the formation of the carcinogenic N-nitrosamines, especially the TSNA in the smoke. However, all analytical measures of the smoke components have been established on the basis of standardized machine smoking conditions, such as those introduced by the Federal Trade Commission, that call for 1 puff to be taken once a minute over a 2-s period with a volume of 35 ml. These smoking parameters may have simulated the way in which people used to smoke the high-yield cigarettes; however, they no longer reflect the parameters applicable to contemporary smokers, and especially not those applicable to the smoking of low- and ultra-low-yield filter cigarettes. Recent smoking assays have demonstrated that most smokers of cigarettes with low nicotine yield take between 2 and 4 puffs per minute with volumes up to 55 ml to satisfy their demands for nicotine. The overview also discusses further needs for reducing the toxicity and carcinogenicity of cigarette smoke. From a public health perspective, nicotine in the smoke needs to be lowered to a level at which there is no induction of dependence on tobacco.

Benzopyrenes

Radiation with 1 Gy prevents the activation of the mitotic inducers mitosis-promoting factor (MPF) and cdc25-C in HeLa cells.

The mechanism of the transient G2 arrest induced by small doses of ionizing radiation involves the failure to activate the correctly formed pre-mitosis-promoting factor (MPF) complex of cyclin B and p34cdc2 by dephosphorylation at Tyr15 of the latter, as recent studies of other laboratories have indicated. Similar data were obtained with the G2 arrest-inducing agents epidermal growth factor and the phorbol ester 12-0- tetradecanoylphorbol-13-acetate (H. Barth and V. Kinzel, Exp. Cell Res., 212: 383-388, 1994, and H. Barth and V. Kinzel, J. Cell. Physiol., 162: 44-51, 1995). To differentiate the radiation consequences in synchronized HeLa cells from those of 12-0-tetradecanoylphorbol-13-acetate and epidermal growth factor, experiments with a very small dose (1 Gy) have been carried out in cells close to the G2-M border and, for comparison, in mitotic cells. We show that in addition to the failure of p34cdc2 dephosphorylation at Tyr15, radiation with 1 Gy also prevents the activation of the phosphatase cdc25-C, the enzyme catalyzing the MPF activation. In contrast, irradiation of mitotic cells with 1 Gy did not influence that fraction of either MPF or cdc25-C already activated. Moreover, the gain in MPM-2 antigenicity of cdc25-C, usually indicative of an activating phosphorylation, is shown to be prevented by 1 Gy. The data indicate that the initiation of the proposed autocatalytic loop between MPF and cdc25-C becomes interrupted by radiation, but they give no hint at which point.

CDC2 Protein Kinase

Role of cdc25-C phosphatase in the immediate G2 delay induced by the exogenous factors epidermal growth factor and phorbolester.

Studies on the link between cellular signalling and cell cycle control at the G2 checkpoint have shown that, in HeLa cells, epidermal growth factor (EGF) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly inhibit the G2-M transition by preventing the key component of mitosis-promoting factor (MPF), p34cdc2, from expressing protein kinase activity. The kinase activity of active MPF is not inhibited; rather, the conversion of pre-MPF to MPF, i.e., the activating dephosphorylation of p34cdc2, at tyrosine is rapidly blocked (Barth and Kinzel, 1994, Exp. Cell Res. 212:383-388; 1995, J. Cell. Physiol., 162:44-51). The phosphatase responsible, cdc25-C, is activated by phosphorylation in mitotic cells starting at the G2-M transition in an autocatalytic loop with MPF (Hoffmann et al., 1993, EMBO J. 12:53-63). We now show that, concomitant with the prevention of MPF activation, EGF and TPA induced a reduction of the activity of cdc25-C in synchronized cultures. Furthermore, treatment of mitotic HeLa cells with TPA did not influence the kinase activity of MPF but caused a rapid decrease of the specific enzyme activity of cdc25-C, probably due to dephosphorylation of the enzyme, as indicated by reduced binding of monoclonal MPM-2 antibody specific for phosphoepitopes in M phase. Because of its inability to induce signalling during division, EGF failed to influence the activity of cdc25-C in mitotic cells. The scenario in cells late in G2 that are committed to enter mitosis may be as follows: In those cells where the signalling pathways responding to EGF as well as those responding to TPA are still open, cdc25-C is prevented by dephosphorylation from exceeding the threshold level of activity required to initiate the activation of and the autocatalytic feedback loop with p34cdc2 and to enter mitosis. Therefore, cdc25-C appears to represent part of an interface between cellular signalling and cell cycle control in G2 phase.

CDC2 Protein Kinase

Cloning and expression analysis of a novel mesodermally expressed cadherin.

Cadherins are calcium-dependent cell adhesion molecules which show developmental and tissue-specific expression. Here we report the cloning of a mouse cadherin which is predominantly expressed in tissues of mesodermal origin. In contrast to other cadherins, cadherin-11 expression is largely restricted to mesenchymal tissues surrounding various organs but is not found in epithelia. Sequence analysis suggests that this cadherin is the mouse homologue of the previously reported human cadherin-11 and is a member of a cadherin subfamily, which is evolutionarily distinct from other cadherin subfamilies identified so far.

Amino Acid Sequence

Effect of phenylarsine oxide on the fission yeast Schizosaccharomyces pombe cell cycle.

Phosphotyrosyl turnover is an essential regulatory mechanism for many biological processes, and the balance between tyrosine kinases and phosphatases plays a major role in the control of cell proliferation. Phenylarsine oxide (PAO), a potent inhibitor of tyrosine phosphatases (PTPase), was used to investigate the involvement of PTPase in the growth and control of the cell cycle of the fission yeast Schizosaccharomyces pombe. Cell proliferation was arrested by treatment with PAO, which was found to inhibit cdc25 PTPase in vitro but appeared not to act in vivo on this mitosis inducer. The PAO-treated cells displayed a mono- or binucleated phenotype and a DNA content that was either 2C or 4C, indicating a cell cycle arrest with a failure to complete cytokinesis. Entry into the cell division cycle from the G0 quiescent stage was also delayed by treatment with PAO. These results suggest that a number of key events in the mitotic cell cycle are regulated by as yet unidentified PTPases.

Arsenicals

Plasma chromogranin A marks emesis and serotonin release associated with dacarbazine and nitrogen mustard but not with cyclophosphamide-based chemotherapies.

Chromogranin A (CgA) is present in high concentrations in enterochromaffin cells, where it is co-localised with serotonin in the storage granules. Plasma CgA has been reported to mark emesis and serotonin release associated with cisplatin treatment. However, it is not known whether plasma CgA could be an indicator of emesis and of serotonin release in patients receiving non-cisplatin chemotherapies. Therefore, in this study we evaluated, in cancer patients, the temporal relationships between the increases in plasma CgA and urinary 5-hydroxyindoleacetic acid (5-HIAA) and the development of vomiting following dacarbazine, nitrogen mustard and cyclophosphamide treatments. Metoclopramide was used as antiemetic. With dacarbazine, nitrogen mustard and cyclophosphamide the median time to the onset of emesis was 2.3, 2.8 and 5.3 h and the duration of intense emesis was 3, 2 and 6 h respectively. Plasma CgA and urinary 5-HIAA increased after dacarbazine- and nitrogen mustard-based chemotherapies, with maximal increases between 4 and 6 h after initiation of drug infusion. The time course for the increases in plasma CgA paralleled that of urinary 5-HIAA and the period of intense emesis. A highly significant (P = 0.0009) positive correlation (r = 0.68) was found between the increases in plasma CgA and in urinary 5-HIAA. Cyclophosphamide treatment was not associated with increases in plasma CgA and in urinary 5-HIAA, despite inducing emesis; this indicates that the increases in CgA and 5-HIAA after dacarbazine and nitrogen mustard are not due to the act of vomiting per se. In summary, plasma CgA is a marker of serotonin release (most likely from enterochromaffin cells) after dacarbazine and nitrogen mustard-based chemotherapies, exocytosis being the most likely mechanism for the release of serotonin. Serotonin released from enterochromaffin cells seems to trigger the emetic response to dacarbazine and nitrogen mustard; however, cyclophosphamide may release serotonin from a different pool (enteric serotonin neurons and/or CNS serotonin?).

Adult

Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard.

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.

Aphidicolin

Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.

Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.

Bisbenzimidazole

The role of cdc25 in checkpoints and feedback controls in the eukaryotic cell cycle.

Major checkpoints that gate progression through the cell cycle function at the G1/S transition, entry into mitosis and exit from mitosis. Cells use feedback mechanisms to inhibit passage through these checkpoints in response to growth control signals, incomplete DNA replication or spindle assembly. In many organisms, transition points seem to involve regulation of the activity of cyclin-dependent kinases (cdks) not only through their interactions with various cyclins, but also by phosphorylation-dephosphorylation cycles acting on the kinase activity of the cdks. These phosphorylation cycles are modulated by the regulation of the opposing kinases and phosphatases that act on cdks and form feedback loops. In this article, we discuss the role of positive and negative feedback loops in cell cycle timing and checkpoints, focusing more specifically on the regulation of the dual specificity cdc25 phosphatase.

Animals