Context-dependent adaption of EpCAM expression in early systemic esophageal cancer.

The role of the epithelial cell adhesion molecule EpCAM in cancer progression remains largely unclear. High expression of EpCAM in primary tumors is often associated with more aggressive phenotypes and EpCAM is the prime epithelial antigen in use to isolate circulating tumor cells (CTCs) and characterize disseminated tumor cells (DTCs). However, reduced expression of EpCAM was associated with epithelial-to-mesenchymal transition (EMT) and reports on a lack of EpCAM on CTCs emerged. These contradictory observations might reflect a context-dependent adaption of EpCAM expression during metastatic progression. To test this, EpCAM expression was monitored in esophageal cancer at different sites of early systemic disease. Although most of the primary esophageal tumors expressed high levels of EpCAM, the majority of DTCs in bone marrow lacked EpCAM. In vitro, downregulation of EpCAM expression at the plasma membrane was observed in migrating and invading cells, and was associated with a partial loss of the epithelial phenotype and with significantly decreased proliferation. Accordingly, induction of EMT through the action of TGFß resulted in substantial loss of EpCAM cell surface expression on esophageal cancer cells. Knock-down or natural loss of EpCAM recapitulated these effects as it reduced proliferation while enhancing migration and invasion of cancer cells. Importantly, expression of EpCAM on DTCs was significantly associated with the occurrence of lymph node metastases and with significantly decreased overall survival of esophageal cancer patients. We validated this observation by showing that high expression of EpCAM promoted tumor outgrowth after xenotransplantation of esophageal carcinoma cells. The present data disclose a dynamic expression of EpCAM throughout tumor progression, where EpCAM(high) phenotypes correlate with proliferative stages, whereas EpCAM(low/negative) phenotypes associated with migration, invasion and dissemination. Thus, differing expression levels of EpCAM must be taken into consideration for therapeutic approaches and during clinical retrieval of disseminated tumor cells.

EpCAM regulates cell cycle progression via control of cyclin D1 expression.

The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway.

Species variation in the testicular angiotensin converting enzyme promoter studied in transgenic mice.

We have studied the control of transcription of the testicular angiotensin converting enzyme (ACEt) in normal and transgenic mice. Northern analyses, including a developmental curve and separated germ cells, for ACEt mRNA suggest predominantly post-meiotic expression. Mice transgenic for a construct containing the proximal 298 bp of the rabbit ACEt promoter, with chloramphenicol acetyl transferase (CAT) as a recorder, showed correct tissue regulation while a 86 bp fragment of the promoter led to no expression. Many candidate transacting factor binding elements, previously identified as candidate regulators of transcription driving spermatogenesis, are scattered across this 298 bp in the rabbit (but not the mouse) promoter and may lead to tissue specificity. The recent finding that the proximal 91 bp of the mouse ACEt promoter leads to tissue specific expression of a recorder gene (Howard et al., 1993) emphasizes the difference between the two species and the importance of a cAMP response element (CRE) within this fragment for tissue specific expression. This CRE is conserved in the rabbit promoters we used.

The structures of the bovine and porcine proacrosin genes and their conservation among mammals.

Sperm acrosin is a serine protease that is involved in the recognition, binding and penetration of the sperm of the zona pellucida of the ovum. The bovine and porcine genes were cloned and characterized. Alignment of the intron/exon structure of both genes with the previously characterized human, rat and mouse genes and with other serine protease genes reveals that the coded sequence of the mammalian proacrosin is distributed in 5 exons and the splice junction types are identical to the exons encoding the catalytic domain of other serine protease genes. A comparison of the bovine, porcine, human, guinea pig, rabbit, rat and mouse preproprotein sequences shows that the catalytic domain is highly conserved, while the sequence of the proline rich domain is very variable among the species, ranging from 28.9% to 68.8%.

[Prognosis in gynecologic diseases]. / Zur Prognose gynäkologischer Erkrankungen.

Besides early diagnosis, the prognosis of gynecological disease is dependent on the state of the urinary tract. Mainly tumours and site anomalies of the female genital system as well as endometriosis and chronic-inflammatory masses of the appendages lead to malfunction of urinary bladder, ureter and kidneys. Ureteric changes are seen in 3 to 55% of genital tumours and in 20 to 40% of cases of endometriosis. Ureteric and kidney dysfunction can occur even more often in longstanding site anomalies. Typical renal complications of gynecological diseases are pyelonephritis, pyelectasy and hydronephrosis. Their frequency lies between 8 and 40% depending on kind and duration of the underlying disease. The figures shown, indicate that renal and ureteric changes sometimes are more serious than the genital disease. Improvement of prognosis can only be achieved by early recognition of the underlying gynecological disease together with the timely proof of renal and ureteric dysfunction.

Stage and developmental specific gene expression during mammalian spermatogenesis.

Spermatogenesis is a complex developmental process which involves amplification of germinal stem cells, their differentiation into spermatocytes, meiotic division and finally transformation into mature spermatozoa. Therefore, spermatogenesis provides an interesting system for examining the regulation of gene expression during development and differentiation. The genes expressed during spermatogenesis can be divided into two main groups: diploid and haploid expressed genes. In this review, we report about the regulation of expression of a diploid expressed gene, namely the proacrosin gene, and that of a haploid expressed gene, the transition protein 2 gene.

Cloning, expression, and chromosomal localization of the rat mitochondrial capsule selenoprotein gene (MCS): the reading frame does not contain potential UGA selenocysteine codons.

The mitochondrial capsule selenoprotein (MCS) is a selenium-containing polypeptide. It is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. In this paper, we report the isolation and characterization of the rat MCS cDNA and gene. The cDNA contains a reading frame for a 145-amino-acid protein and it lacks the UGA codons, which have been found in the reading frame of the mouse MCS cDNA and have been presumed to encode the selenocysteine in the amino terminal of the deduced mouse amino acid sequence. The deduced amino acid sequence of the rat and mouse MCS shows a high level of homology (79%). The rat MCS gene contains two exons; the intron sequence interrupts the 5' untranslated sequence at the same position as in the mouse MCS gene. The transcription start site is located 184 bp upstream of the translation start site. Alignment of the 5'-flanking regions of the mouse and rat genes reveals that the first 400 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 73%. This conserved region contains no TATA or CAAT box motifs. Northern blot analysis indicates that the MCS mRNA is detectable only in the testis after day 30 of postnatal development. Moreover, in situ hybridization revealed that the rat MCS gene is mainly expressed in round spermatids. From the analysis of mouse-rat cell hybrids that segregate rat chromosomes, the MCS gene was assigned to rat chromosome 2.

[Labor-induced bladder injuries: historical observations]. / Geburtsbedingte Blasenverletzungen: Historische Betrachtungen.

Injuries to the urinary bladder with development of a fistula during birth were first mentioned around 1030 AD in the opus called 'Al-Kanoun' by the Arabic physician and philosopher Avicenna (Ali Ibn Sina). The observations of D.E. Derry in the mummy of Henhenit seem to have made sure that this obstetric complication already existed earlier on. Henhenit lived at the court of king Mentuhotep II (around 2050 BC). During the second half of the 19th century injury-related and necrosis-related fistulas were distinguished for the first time. Jobert de Lamballe (1852), Marion Sims (1852), and Gustav Simon (1854) created the basis for successful operative treatment of vesicovaginal fistulas.

Molecular cloning and characterization of the bovine and porcine outer dense fibers cDNA and organization of the bovine gene.

Outer dense fibers (ODF) or accessory fibers are filamentous structures of the sperm tail of many eumetozoan organisms endowed with internal fecundation. The bovine and porcine cDNA of an outer dense fiber protein was cloned, sequenced and compared to the previously characterized human and rat cDNA sequences. The coding sequences and the 5' and 3' untranslated regions of the ODF cDNAs are highly conserved. A comparison of the bovine, porcine, human and rat ODF protein sequences revealed that the protein displays a high degree of similarity, ranging from 87% to 98%. The ODF protein is rich in cysteine and contains the C.X.P. repeat at the C-terminal which is different in number among mammalian species. All the 27 cysteine residues in the ODF sequence except those in the C.X.P. repeat are conserved in the four species. We report here also the organization of the bovine ODF gene which is similar to that of human and rat. The transcription start site in the bovine ODF gene is localized 98 bp upstream of the translation start site. Alignment of the 5' flanking region of bovine ODF with the rat gene reveals that the first 130 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 83%. This conserved region contains a TATA-like box (TTTAAA) and binding sites for AFT/CREB and EGR-1 transcription factors.

[Expert assessment of gynecologic diseases]. / Begutachtung gynäkologischer Erkrankungen.

Medical reports in Gynecology have to consider the evaluation of organic diagnosis as well as connected psychical disorders. It has to be examined if psychical insults have led to dysfunction in the genital system. A typical example is hypothalamic amenorrhea as a sequel of extreme stress or chance of surroundings. Frequent lesions in the urinary system due to endometriosis, change of site, tumours and chronic inflammation of the female genital system often require a urological as well as a gynecological examination. Severity and duration of complaint, tendency to recur and the necessity of clinical treatment are essential factors in the assessment of the degree of disability. Examples of medical reports are shown and the limits of modified professional discretion are pointed out.

DNA-protein binding studies in the 5' flanking region of rat proacrosin gene which is transcribed in diploid germ cells.

The proacrosin gene is transcribed in diploid spermatogenic cells and translated in haploid round spermatids. In order to evaluate sequences which are involved in proacrosin gene transcription, DNA-protein interactions were analyzed in 1.2 kb of the 5'flanking region of the rat gene. 13 protein binding sites were identified by DNase I footprinting using nuclear extracts from rat testis and brain, respectively. Five footprints (F1, F3, F7, TS2, TS3) which suggest an interaction with testis specific nuclear factors were further examined by gel retardation assays. Three testis specific binding sites (F1, F7, TS2, located 472bp, 697bp and 1004bp upstream of ATG, respectively) could be identified with both methods. The binding site F1 contains a motif which is similar to a testis specific footprint found in mouse protamine 1 gene. The nucleotide sequence of F7 contains the recognition motif of an isoform of the transcription factor GATA1, which is expressed in testis. Furthermore F1 and F7 are located in that part of the 5'flanking region of the proacrosin gene, which can direct proacrosin gene expression in germ cells of male transgenic mice.

Functional and molecular characterization of the transcriptional regulatory region of the proacrosin gene.

Proacrosin, the zymogen form of the serine protease acrosin, is located within the acrosomal vesicle of mammalian spermatozoa and has been suggested to be involved in the fertilization process. In mouse and rat, expression of the proacrosin gene starts in pachytene spermatocytes and continues through the early stages of spermiogenesis. We have shown recently that 2.3 kilobase pairs of the 5'-flanking region of the rat proacrosin gene is sufficient to direct chloramphenicol acetyltransferase gene expression in a germ cell-specific and developmental stage-specific manner in the mouse. Additional transgenic lines have been generated which include two deletions in the 5'-flanking region and a tyrosinase minigene as marker for gene expression. Transgenic mice bearing these two truncated fragments showed different patterns of reporter gene expression. Transgenic lines (BM, B3, B2) harboring the 397-base pair (bp) fragment (from 45 to 442 bp upstream of ATG) showed no chloramphenicol acetyltransferase (CAT) activity in either testis or other tissues, but analysis via reverse transcription polymerase chain reaction confirmed low levels of reporter gene transcription in testis. Transgenic line TC bearing a longer fragment of 877 bp (from 45 to 922 bp upstream of ATG) showed a reporter gene expression and chloramphenicol acetyltransferase enzyme activity which was identical to that found in mice harboring the 2.3-kilobase pair 5'-flanking region. The analysis of the CAT gene expression during testicular development showed diploid transcription and haploid translation. It can be concluded that all sequences required for a basic level of testis-specific transcription of transgene are present within the 397-bp fragment, and other DNA sequences located outside of the 397-bp fragment but present within the 877-bp fragment can function as enhancer elements. Two fragments within the 877-bp region were identified by gel retardation assays as binding exclusively to nuclear factor(s) from testis protein extracts. In both fragments we identified sequence elements which are present in the promoter region of the germ cell-specific genes for histone H2B and protamine 1, respectively.