Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein.

AIM: Von Hippel-Lindau protein (VHL) provides the degradation of hypoxia-inducible factor (HIF). Tetracycline-induced, Pax8-rtTA-based knockout of VHL (VHL-KO) affects all renal tubules and periportal hepatocytes and leads to sustained upregulation of HIF. Here, we study the phenotype of VHL-KO in both organs, the time course of changes, and long-term morpho-functional outcome. METHODS: Mice with doxycycline-induced VHL-KO and controls (CON) were followed for up to 9 months. Systemic and tissue parameters were evaluated using clinical chemistry, histology, immunohistochemistry, RT-PCR and in situ hybridisation. RESULTS: At day 3 following VHL-KO, substantial abundance of HIF-1α and -2α was detected in the nuclei of hepatocytes and renal tubular epithelia. Hypoxia, induced by bleeding anaemia, did not further augment HIF signal. Erythropoietin mRNA was detectable in hepatocytes but not in the kidney. Vascular endothelial growth factor mRNA was upregulated in kidney but not in liver. At day 7 following VHL-KO, the renal capillary density was enhanced, reaching its maximum at day 14. Blood haemoglobin increased constantly up to day 28 (23.3 vs. 15.8 g dL(-1) , VHL-KO vs. CON). Thereafter, it was kept within the normal range by weekly blood collections. Pathological changes were absent from kidney and liver 9 months after VHL-KO. CONCLUSIONS: Inducible, Pax8-rtTA-based deletion of VHL leads to organ-specific expression of epithelial HIF and erythropoietin in liver and kidney without causing pathological changes. Uniform, maximal and sustained HIF activation along the renal tubule may serve to study the potential benefits of hypoxia adaptation in experimental renal injury.

Predominant mutation of codon 41 of the beta-catenin proto-oncogene in rat colon tumors induced by 1,2-dimethylhydrazine using a complete carcinogenic protocol.

Constitutive activation of the wnt-signaling pathway plays an important role during both human and rat colon carcinogenesis and can be brought through mutations in either the adenomatous polyposis coli or the beta-catenin gene. Mutations found in the beta-catenin gene typically affect one out of four regulatory phosphorylation sites near the N-terminus of the beta-catenin protein. Whereas in human colon cancers, however, the majority of beta-catenin mutations directly alter threonine 41 or serine 45; the beta-catenin mutations found in chemically induced rat colon tumors seemed to cluster around codon 33 instead. Unlike previous studies, that have used relatively short-term (2-5 weeks) treatment with one of the alkylating agents 1,2,-dimethylhydrazine (DMH) or azoxymethane, we have investigated the mutational spectrum of the beta-catenin gene in a panel of rat colon tumors induced by long-term (20 weeks) DMH-treatment. We detected beta-catenin mutations in 12 of 33 (36%) tumors. Interestingly, only one of the beta-catenin mutations found affected the previously implicated codon 33 cluster region (Asp32Asn), whereas 11 of 12 (>90%) mutations represented identical C-->T transitions within codon 41 resulting in the common replacement of threonine by isoleucine. We propose a model in which codon 41 mutations bear higher oncogenic potential but are induced by DMH less frequently than mutations in the codon 33 cluster region. Consequently, only after sustained carcinogenic treatment, as is achieved in the long-term DMH-protocol, codon 41 mutations will be induced frequently enough to be present in all developing malignant lesions and, then, because of their higher oncogenic potential, these are selected for.

Human eukaryotic initiation factor EIF2C1 gene: cDNA sequence, genomic organization, localization to chromosomal bands 1p34-p35, and expression.

We report the cloning and characterization of the human eukaryotic protein translation initiation factor EIF2C1 gene. The human EIF2C1 gene consists of 19 exons and 18 introns that span a region of almost 50 kb. It is located on the short arm of chromosome 1 in the region 1p34-p35. This genomic region is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. The human EIF2C1 gene is ubiquitously expressed at low to medium levels. Differential polyadenylation and splicing result in a complex transcriptional pattern. The cDNA sequence is 7478 bp long and contains an extremely large 3' untranslated region of 4799 bp with multiple, short repeated segments composed of mono-, tri-, or quattronucleotides interspersed throughout. The human EIF2C1 gene belongs to a multigene family in human. It is highly conserved during evolution, sharing about 90% identity with rabbit eIF2C and 70% identity with plant AGO1 at the amino acid level. These facts suggest that human EIF2C1 might play an important physiological role.

Mutational activation of the beta-catenin proto-oncogene is a common event in the development of Wilms' tumors.

Activation of beta-catenin-mediated transcription is the nuclear end point of organ-specific Wnt signaling. In the developing kidney, Wnt-4, a secreted glycoprotein, acts as an autoinducer of the mesenchymal to epithelial transition that underlies normal nephron development. Dysregulation of this epithelial transformation process may lead to Wilms' tumors (WTs). In this study, we investigated the potential role of the beta-catenin proto-oncogene, a candidate downstream target molecule of Wnt-4 signaling, in the development of WTs. In 6 of 40 tumors (15%), mutation analysis revealed heterozygous missense mutations or small deletions that result in the loss of important regulatory phosphorylation sites within the beta-catenin protein. These findings indicate that activating beta-catenin mutations may play a significant role in the development of WTs and establish a direct link between Wilms' tumorigenesis and the Wnt signal transduction pathway governing normal kidney development.

Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pI of 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescence in situ hybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the "fused gene" subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.

Routine karyotyping in Wilms tumor.

We describe the karyotypes of nine Wilms tumors (WT). Four tumors were initially karyotyped from diagnostic needle core biopsies, 3 after postchemotherapy tumor resection and the remainder from xenografts grown in nude mice. The 9 nephroblastomas were composed of 7 with favorable histology (intermediate-grade malignancy) and 2 with unfavorable histology (anaplastic or high-grade malignancy). The 7 tumors with favorable histology had karyotypes typical of WT, with the previously described nonrandom abnormalities +1q, +6, +7, +8, +12, +13, +18 and structural abnormalities of 1p and 16q present in at least 1 case. The most common abnormalities were trisomy 18 (4 cases) and +1q (3 cases). The 2 tumors with unfavorable histology both had complex karyotypes atypical for WT. We suggest that cytogenetics can act as a marker when histologic grade is in doubt. Karyotypic analysis from needle core biopsies was attempted in 6 samples, including 1 from a nephrogenic rest (NR) of the nonaffected kidney and provided a result on 5 occasions. The NR were present in the sole case with a constitutional abnormality, a mosaic partial duplication of 8q. However, both the tumor and the NR were apparently derived from the normal cell line. Here we demonstrate that a cytogenetic result can be routinely obtained from needle core biopsies and will thus facilitate true diagnostic tumor karyotypes in both WT and other tumors.

Polymerase chain reaction (PCR) detection of porcine Chlamydia trachomatis and ruminant Chlamydia psittaci serovar 1 DNA in formalin-fixed intestinal specimens from swine.

In previous studies chlamydiae were detected immunohistologically in the gut of 66 out of 311 pigs. The aim of the present investigation was the classification of these intestinal porcine chlamydiae. For the study, DNA extracted from 52 paraffin-embedded intestinal tissues was amplified in nested polymerase chain reactions (PCRs) with Chlamydia omp1 genus- and species-specific primers. Some of the amplification products were cloned and sequenced. In 45 cases DNA could be amplified with genus-specific primers. Species-specific PCR and sequencing showed that in 42 cases the chlamydial omp1 genotype was Chlamydia trachomatis. Sequenced DNA fragments were 95-99% identical with the porcine strain S45. In three further cases sequencing analysis provided DNA sequences which were 100% identical with Chlamydia psittaci B577 (serovar 1) omp1 genotype. So far as the authors are aware this is the first report on the occurrence of C. psittaci serovar 1 in pigs.

Mixed infections with porcine Chlamydia trachomatis/pecorum and infections with ruminant Chlamydia psittaci serovar 1 associated with abortions in swine.

In a previous immunohistological study, chlamydiae were detected in 5 out of 139 cases of swine abortion, and a possible implication of C. psittaci serovar 1 was suggested. The present study sought to classify the chlamydiae found in the fetal organs of these abortions. DNA extracted from 15 paraffin-embedded tissue specimens (10 livers and 5 lungs, obtained from 10 fetuses from 9 cases of abortion) was amplified in a nested PCR with Chlamydia omp1 genus-specific primers. Chlamydia DNA was amplified in 9 liver and 2 lung specimens. Eight of the amplification products were cloned, and 5 clones of each amplification were sequenced. Sequence analysis demonstrated in 7 specimens the simultaneous presence of porcine C. trachomatis S45 and C. pecorum 1710S omp1 genotypes. All DNA fragments of 1 amplification were identical to the ruminant C. psittaci B577 omp1 genotype (serovar 1). The results suggest that mixed infections with porcine C. trachomatis and C. pecorum dominate chlamydial infections associated with abortion in swine, but ruminant abortigenic C. psittaci are also found.