Henrik Leffers

Emerging endocrine disrupters: perfluoroalkylated substances

In recent years, polyfluorinated chemicals (PFCs) have increasingly been used as surfactants in various industry- and consumer products, because of their unique properties as repellents of dirt, water and oils. The most well-known PFCs are perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and their derivatives belonging to the group of perfluoroalkylated substances. The PFCs are very persistent in the environment, and some of them have been discovered as global pollutants of air, water, soil and wildlife and even found in remote polar areas. Bioaccumulation occurs also in humans, and everybody in our society has traces of these PFCs in their blood and internal organs such as the liver, kidneys, spleen, gall bladder and testes. In the blood, PFOS and PFOA are bound to serum proteins. The acute toxicity of the polyfluorinated substances is moderate but some substances can induce peroxisome proliferation in rat livers and may change the fluidity of cell membranes. Some of these PFCs, such as PFOS and PFOA, are potential developmental toxicants and are suspected endocrine disruptors with effects on sex hormone levels resulting in lower testosterone levels and higher oestradiol level. Other PFCs have oestrogenic effects in cell cultures. The industrial production of PFOS and its derivatives stopped in 2000, and the European Union has banned most uses from the summer of 2008. However, hundreds of related chemicals: homologues with shorter or longer alkyl chain, PFOA and telomers, which potentially may degrade to perfluoroalkanoic (carboxylic) acids, are not regulated.

Transcription Factor AP-2γ Is a Developmentally Regulated Marker of Testicular Carcinoma In situ and Germ Cell Tumors

Purpose: Transcription factor activator protein-2γ (TFAP2C, AP-2γ) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2γ in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2γ and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia. Experimental Design: We analyzed expression pattern of AP-2γ at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2γ in normal and dysgenetic samples. We also investigated the regulation of AP-2γ by steroids and retinoic acid. Results: We detected abundant AP-2γ in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2γ in somatic tumors. We found that AP-2γ expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2γ protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2γ was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells. Conclusions: AP-2γ is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2γ is a novel marker of testicular CIS and CIS-derived tumors.

High-resolution comparative genomic hybridization detects extra chromosome arm 12p material in most cases of carcinoma in situ adjacent to overt germ cell tumors, but not before the invasive tumor development.

High‐resolution comparative genomic hybridization (HR‐CGH) analysis was performed on DNA purified from laser‐capture microdissected carcinoma in situ (CIS) cells from nine cases of CIS, either from tissue without any invasive tumor or from testicular parenchyma adjacent to seminoma, nonseminoma, or a combined germ cell tumor. Before CGH analysis, DNA was amplified by degenerate oligonucleotide primed PCR (DOP‐PCR) and directly labeled with a mixture of FITC‐dUTP and FITC‐dCTP. CGH analysis revealed extra chromosome arm 12p material in six out of seven cases with CIS adjacent to overt tumors, but only a diminutive gain of 12q was noted in one of the two cases of CIS without invasive elements. In addition, gains of parts of chromosome 8 (3/7) and losses of chromosome 5 (2/7) were demonstrated in CIS adjacent to invasive tumors. Gains of parts of chromosome 7 were found in CIS adjacent to seminoma (4/4), whereas relative gains of chromosome 15 were identified in some cases of CIS adjacent to seminoma and in isolated CIS in comparison to CIS adjacent to nonseminoma. Our data seem to indicate that extra 12p material is not present in the “dormant” CIS cell before development of an invasive tumor. The gain of extra chromosome 12 material may not be an early event in the neoplastic transformation, but is most likely associated with a more malignant progression of the CIS cell.

Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms

BACKGROUND UTF-1 and REX-1/ZFP42 are transcription factors involved in pluripotency. Because of phenotypic similarities between pluripotent embryonic stem cells and testicular germ cell tumours (TGCT) and the derivation of pluripotent cells from testes, we investigated the expression of UTF-1 and REX-1 during human gonadal development and in TGCT. METHODS Expression of UTF-1 and REX-1 was studied in 52 specimens from human gonadal development and in 86 samples from TGCT. RESULTS UTF-1 and REX-1 were expressed throughout male gonadal development. In the mature testis, UTF-1 was expressed in spermatogonia, whereas REX-1 was expressed in meiotic cells and, together with OCT-3/4, in primary oocytes. Both UTF-1 and REX-1 were expressed in testicular carcinoma in situ and in TGCT. Contrarily to REX-1, UTF-1 was expressed in all spermatocytic seminomas. CONCLUSIONS Unlike other pluripotency markers NANOG and OCT-3/4, UTF-1 and REX-1 are expressed throughout human testes development. The expression pattern indicated that UTF-1 plays a possible role in spermatogonial self-renewal, whereas expression of REX-1 in meiotic cells from both testes and ovary indicate a role in meiosis. UFT-1 and REX-1 are expressed in TGCT and the high abundance of UTF-1 in spermatocytic seminomas is consistent with the hypothesis that this tumour type originates from spermatogonia.

Cloning and expression of a novel human profilin variant, profilin II

We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kidney and less strongly in heart, placenta, lung and liver. In addition, three different transcript lengths were detected. Only one transcript of profilin I was found. The expression level of this was low in brain and skeletal muscle, medium in heart and high in placenta, lung, liver and kidney.

Human cellular protein patterns and their link to genome DNA sequence data: Usefulness of two-dimensional gel electrophoresis and microsequencing

Analysis of cellular protein patterns by computer‐aided 2‐dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2‐dimensional gel protein databases that may link protein and DNA information and that offer a global approach to the study of the cell. Using the integrated approach offered by 2‐dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2‐dimensional gel protein databases are becoming increasingly important in view of the concerted effort to map and sequence the entire genome.—Celis, J. E.; Rasmussen, H. H.; Leffers, H.; Madsen, P.; Honoré, B.; Gesser, B.; Dejgaard, K.; Vandekerckhove, J. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two‐dimensional gel electrophoresis and microsequencing. FASEB J. 5: 2200–2208; 1991.

Testicular dysgenesis syndrome and the origin of carcinoma in situ testis

Recent increases in male reproductive disorders have been linked to exposure to environmental factors leading to the testicular dysgenesis syndrome (TDS). Testicular cancer is the most severe condition in TDS and studies have shown a clear correlation between risk of testicular cancer and other components of TDS and that the geographical location of the mother during pregnancy can be a risk factor. This suggests that the dysgenesis has its origin in utero and that TDS is initiated by environmental factors, including possibly hormone-disrupting compounds that act on the mother and the developing foetus, but the genetic background may also play a role. The morphological similarity of carcinoma in situ (CIS) cells (the precursor of the majority of invasive testicular cancers) with primordial germ cells and gonocytes, and overlap in expression of protein markers suggests an origin of CIS from primordial germ cells or gonocytes. CIS cells and germ cell-derived cancers of the human type have so far not been described in any animal model of TDS, which could be caused by species differences in the development of the male gonad. Regardless of this, it is plausible that the dysgenesis, and hence the development of CIS cells, is a result of disturbed signalling between nurse cells and germ cells that allow embryonic germ cells to survive in the pre-pubertal and adult testis. The post-pubertal proliferation of CIS cells combined with aberrant signalling then leads to an accumulation of genetic changes in the CIS cells, which eventually results in the development of invasive testicular cancer in the adult.

MicroRNA expression profiling of carcinoma in situ cells of the testis

Testicular germ cell tumours, seminoma (SE) and non-seminoma (NS), of young adult men develop from a precursor cell, carcinoma in situ (CIS), which resembles foetal gonocytes and retains embryonic pluripotency. We used microarrays to analyse microRNA (miRNA) expression in 12 human testis samples with CIS cells and compared it with miRNA expression profiles of normal adult testis, testis with Sertoli-cell-only that lacks germ cells, testis tumours (SE and embryonal carcinoma (EC), an undifferentiated component of NS) and foetal male and female gonads. Principal components analysis revealed distinct miRNA expression profiles characteristic for each of the different tissue types. We identified several miRNAs that were unique to testis with CIS cells, foetal gonads and testis tumours. These included miRNAs from the hsa-miR-371-373 and -302-367 clusters that have previously been reported in germ cell tumours and three miRNAs (hsa-miR-96, -141 and -200c) that were also expressed in human epididymis. We found several miRNAs that were upregulated in testis tumours: hsa-miR-9, -105 and -182-183-96 clusters were highly expressed in SE, while the hsa-miR-515-526 cluster was high in EC. We conclude that the miRNA expression profile changes during testis development and that the miRNA profile of adult testis with CIS cells shares characteristic similarities with the expression in foetal gonocytes.

A ribosomal RNA operon and its flanking region from the archaebacterium Methanobacterium thermoautotrophicum, marburg strain: transcription signals, RNA structure and evolutionary implications

Summary One of the two ribosomal RNA operons of the thermophilic methanogen Methanobacterium thermoautotrophicum was sequenced and examined for RNA transcriptional signals and limits, gene organization and the primary and secondary structure of the 16S RNA and 5 S RNA. One transcriptional start was identified 213 nucleotides upstream from the 16S RNA gene, preceded by a long T-rich sequence that could be a promoter. Two other sites were identified 159 and 91 nucleotides upstream that are either both transcription starts or both processing sites. A termination site was localized downstream from the 5S RNA gene at the end of a run of pyrimidines. Both 16S RNA and 5S RNA generate universal secondary structures with G-C contents no higher than those of the non-thermophilic methanogens. Open reading frames border the ribosomal RNA operon that lack Shine-Dalgarno sequences and exhibit no homology with those of known open reading frames bordering other ribosomal RNA operons. Phylogenetic analyses of the 16S RNA sequences confirm that the methanogens constitute a sub-group of the archaebacteria and that M. thermoautotrophicum is closely related to M. formicicum .

Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

BackgroundThe amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined.ResultsCyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration.ConclusionsThe present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus.