Günther Weber

Antimicrobial protein hCAP18/LL-37 is highly expressed in breast cancer and is a putative growth factor for epithelial cells

Human cathelicidin antimicrobial protein hCAP18/LL‐37 is an effector molecule of the nonspecific innate immune system. hCAP18/LL‐37 is present in leukocytes and is expressed in skin and other epithelia, where it is upregulated in association with inflammation and injury. In addition, antimicrobial proteins including cathelicidins have been proposed to play a role in the nonspecific defense against tumors. To assess its potential role in tumor host defense, we investigated the expression of hCAP18/LL‐37 in a series of breast carcinomas. Unexpectedly, we found that hCAP18/LL‐37 was strongly expressed in the tumor cells and not in the adjacent stroma. To test the hypothesis that hCAP18/LL‐37 may provide a growth advantage for the tumor cells, we treated human epithelial cell lines with synthetic biologically active LL‐37 peptide and found a significant increase in cell proliferation. In addition, transgenic expression of hCAP18 in 2 different human epithelial cell lines resulted in increased proliferation of both cell types. These findings do not support the hypothesis that LL‐37 has an antitumor effect, but rather suggest that hCAP18/LL‐37 may promote tumor cell growth in breast cancer.

A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

Predictive testing for multiple endocrine neoplasia type 1 using DNA polymorphisms.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominantly inherited predisposition to neoplastic lesions of the parathyroids, pancreas, and the pituitary. We have previously located the predisposing genetic defect to the long arm of chromosome 11 by genetic linkage. In this study, 124 members of six MEN1 families, including 59 affected individuals, were genotyped for restriction fragment length polymorphisms with different DNA probes, and the genetic linkage between these marker systems and MEN1 was determined. 13 marker systems (17 DNA probes) were found to be linked to MEN1. These markers are located within a region on chromosome 11 spanning 14% meiotic recombinations, with the MEN1 locus in the middle. Four of the marker systems are on the centromeric side of MEN1, and four on the telomeric side, based on meiotic crossovers. The remaining five DNA probes are closely linked to MEN1, with no crossovers in our set of families. The 13 marker systems can be used for an accurate and reliable premorbid test for MEN1. In most clinical situations it is possible to identify a haplotype of this part of chromosome 11 with the mutant MEN1 allele in the middle. The calculated predictive accuracy is greater than 99.5% if three such marker systems are informative. Therefore, genetic linkage testing can be used for informed genetic counseling in MEN1 families, and to avoid unnecessary biochemical screening programs.

Expression analysis of endogenous menin, the product of the multiple endocrine neoplasia type 1 gene, in cell lines and human tissues

We have investigated the endogenous expression of menin, a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1). Western blot analysis showed strong expression of menin as a 68 kDa protein in all of 7 human and primate cell lines tested. In a panel of 12 fetal human tissue extracts, 68 kDa menin was readily detected in brain cortex, kidney, pituitary, testis and thymus and weakly detected in thyroid. Reproducible bands other than 68 kDa were observed in adrenal and heart, whereas menin was undetectable in liver, lung, pancreas and skin. Analysis of synchronized HeLa cells revealed no variation in the amount or size of menin throughout the cell cycle. Protein expression was compared between lymphoblastoid cell lines from healthy controls and MEN1 patients carrying nonsense mutations on 1 allele. No truncated protein was detected in either cytoplasmic or nuclear fractions in mutation‐carrying cells. The expression level and cellular location of full‐length menin did not differ between cell lines derived from MEN1 patients and healthy donors. This suggests that the wild‐type allele has been up‐regulated in mutation‐carrying cells to compensate for the loss of 1 functional allele. Int. J. Cancer 85:877–881, 2000.

Targeted disruption of the mouse phospholipase C β3 gene results in early embryonic lethality

In order to investigate the biological function of phosphatidylinositol‐specific phospholipase C (PLC) we generated mutant mice by gene targeting. Homozygous inactivation of PLCβ3 is lethal at embryonic day 2.5. These mutants show poor embryonic organization as well as reduced numbers of cells. Identical phenotypes were recorded in homozygous mutants generated from two independently targeted embryonic stem cell clones. Heterozygous mutant mice, however, are viable and fertile for at least two generations. We also showed that mouse PLCβ3 is expressed in unfertilized eggs, 3‐cell and egg cylinder stages of embryos. In conclusion, these results indicate that PLCβ3 expression is essential for early mouse embryonic development.

Molecular Cloning and Expression of a Functionally Different Alternative Splice Variant of Prointerleukin-1α from the Rat Testis1

We report here the characterization of an alternative splice variant of prointerleukin-1α (proIL-1α), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1α (32proIL-1α) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1α). In situ hybridization demonstrated constitutive expression of the splice transcript in the seminiferous tubules. This alternative complementary DNA lacked the fifth exon, harboring the calpain cleavage site essential for generation of mature 17K IL-1α. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1α, but not of 24proIL-1α. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1α to the mature 17K form and secretion, whereas 24proIL-1α remained unprocessed. Both 32proIL-1α and 24proIL-1α showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1α. In cont...

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus.

SummaryMultiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.

Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18/LL-37 during wounding in human skin in vivo

Please cite this paper as: Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18/LL‐37 during wounding in human skin in vivo. Experimental Dermatology 2009.

Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches

We have characterized the cytogenetic alterations of the human embyronal cell line 293 by spectral karyotyping and G-banding analysis. To investigate its genomic stability, we compared the karyotypes of 293 and its daughter line EcR-293. Genotype profiling through short tandem repeats complemented the analysis. While displaying almost identical STR profiles and thus verifying their origin and their close relation, the two lines were remarkably different in their number of chromosomes and setup of aberrant chromosomes. However, the cell lines retained a stable karyotype in long term culture. The establishment of subclones from EcR-293, expressing inducible lacZ or MEN1 transgenes, only added minor changes to the karyotype. Our study shows that the cytogenetic constitution of a clonal cell line of the 293 origin appears to be sufficiently stable. However, care should be taken when comparing the properties of independent 293 lineages, since clonal variations might be substantial.

Upregulation of VEGF-A Without Angiogenesis in a Mouse Model of Dilated Cardiomyopathy Caused by Mitochondrial Dysfunction

Angiogenesis is implicated in a variety of human pathologies and may also play a role in the progression of heart failure. We have studied the expression of members of the vascular endothelial growth factor (VEGF) and the angiopoietin families and their receptors in mice lacking the mitochondrial transcription factor A. These mice lack functional respiratory chain activity in their myocytes and develop dilated cardiomyopathy (DCM) postnatally. We studied the hearts of the knockout mice by in situ hybridization, Western blotting analysis, and immunohistochemistry. VEGF-A mRNA and protein levels were elevated in the myocardium of the knockouts. Levels of the hypoxia inducible transcription factor 1 alpha (HIF1α) and of glyceraldehyde-3-phosphate dehydrogenase transcripts were also increased, whereas those of angiopoietin−1 and −2 were reduced. Despite the striking upregulation of VEGF-A, there was no increase in capillary density in the knockout hearts. This study suggests that a disturbance in angiogenesis may contribute to the pathogenesis of DCM.