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Biomedical subjects

G F Saunders

Publications and source records attributed to G F Saunders.

At least 37 records · Page 2Linked to original sources
PubMed1997

The Wilms' tumor gene WT1 can regulate genes involved in sex determination and differentiation: SRY, Müllerian-inhibiting substance, and the androgen receptor.

Genital abnormalities associated with Wilms' tumors in the WAGR and Denys-Drash syndromes and the failure of the gonads to develop in Wilms' tumor gene (wt1)-homozygous mutant mice suggest that WT1 may also function in sexual development. To elucidate the mechanism of action of WT1 in embryonal sexual development, we examined how the four isoforms of WT1 regulate the transcription of several genes involved in sexual development using cotransfection assays. SRY (the sex-determining region of the Y chromosome) promoter was strongly activated by the WT1 isoforms without the KTS tripeptide, WT1(-)KTS, but was not activated by the WT1 isoforms with the KTS tripeptide, WT1(+)KTS, in all cells tested. The second alternative splicing site, which inserts the tripeptide KTS, alters the DNA binding capability. The MüAdullerian-inhibiting substance (MIS) promoter was strongly repressed by WT1(-)KTS isoforms and more weakly repressed by the WT1(+)KTS isoforms in Sertoli cells but not in HeLa cells. The androgen receptor (AR) promoter was strongly repressed by the WT1(-)KTS isoforms in all cells tested and was more weakly or not repressed by WT1(+)KTS isoforms depending on cell lines. Electrophoretic mobility shift assays showed strong binding by recombinant WT1(-)KTS protein and weaker or no binding by the WT1(+)KTS protein to DNA probes containing WT1 binding sites from these three promoters. The results of these functional and binding assays suggest that WT1 has an important role in regulation of genes involved in embryonal sexual development and that WT1 can function as a transcriptional activator.

Animals↗
PubMed1996

Differential function of Wilms' tumor gene WT1 splice isoforms in transcriptional regulation.

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor that can repress transcription of a number of genes. WT1 mRNA undergoes alternative splicing at two locations, yielding four different mRNA species and protein products. One alternative splice alters the zinc finger region of WT1, resulting in the addition of three amino acids, Lys-Thr-Ser (KTS), between zinc fingers 3 and 4, altering the binding of WT1 to DNA. Here, we show that the WT1 protein with and without the KTS tripeptide can repress transcription from the human full-length WT1 promoter. Repression of transcription by WT1 has been shown to require two WT1 binding sites. We examined WT1 repression of the human minimal WT1 promoter, which contains two potential WT1 binding motifs. WT1 lacking the KTS tripeptide (WT1-KTS) was unable to repress transcription from a minimal WT1 promoter of 104 base pairs, whereas WT1 containing the KTS tripeptide (WT1+KTS) repressed transcription from the minimal promoter. The ability of WT1+KTS to repress transcription where WT1-KTS could not provided a functional assay to define differential WT1 binding motifs based on the presence or the absence of the KTS tripeptides. We present data defining the differential consensus DNA binding motifs for WT1-KTS and WT1+KTS. We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1-KTS and WT1+KTS.

Alternative Splicing↗
PubMed1996

Expression pattern of WT1 and GATA-1 in AML with chromosome 16q22 abnormalities.

WT1 is a tumor suppressor gene that can repress transcription of many growth-factor and growth-factor receptor genes. We quantitated WT1 expression levels in 62 acute myelogenous leukemia (AML) samples and found that 82% strongly expressed WT1. WT1 expression levels are highest in the undifferentiated and granulocytic French-American-British (FAB) subclasses and lower in the monocytic subclasses. WT1 was strongly expressed in normal CD34+ bone marrow (BM) stem cells but only weakly or not expressed in normal mature blood cells. This suggests that WT1 gene expression is associated with immature cells, which have high proliferative capacities. Previous studies of WT1 gene regulation showed that GATA-1 may regulate WT1 expression. To understand the relationship between WT1 and GATA-1 expression in leukemia, we examined the expression pattern of GATA-1 in the cells described above. Overall, AML samples expressed significant amounts of both WT1 and GATA-1. However, AML samples with 16q22 abnormalities, presumably interrupting the core binding factor (CBF) beta gene expressed lower than normal levels of GATA-1 but high levels of WT1. Our data suggest that the transcription factor CBF beta may be important for GATA-1 gene regulation. Thus, WT1 expression varied in different FAB subclasses, and GATA-1 expression was strongly affected by the presence of chromosome 16q22 abnormalities.

Chromosome Aberrations↗
PubMed1995

Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3.

It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.

Animals↗
PubMed1995

Regulation of the proto-oncogenes bcl-2 and c-myc by the Wilms' tumor suppressor gene WT1.

The Wilms' tumor gene WT1 functions as a tumor suppressor gene, repressing transcription of several growth factors and growth factor receptors. The bcl-2 and c-myc proto-oncogenes are essential for regulation of apoptosis and cell proliferation with roles in development and oncogenesis. We found that WT1 can repress transcription of both the bcl-2 and c-myc promoters. This suggests that WT1 regulates bcl-2 and c-myc during renal development, and the loss of functional WT1 results in deregulation of bcl-2 and c-myc, contributing to tumor formation.

Amino Acid Sequence↗
PubMed1995

Transcriptional silencer of the Wilms' tumor gene WT1 contains an Alu repeat.

Expression of the Wilms' tumor gene WT1 is tightly regulated throughout development. In contrast, the WT1 promoter is promiscuous, functioning in all cell lines tested. We have cloned a transcriptional silencer that is involved in regulation of the WT1 gene. The transcriptional silencer is located in the third intron of the WT1 gene, approximately 12 kilobases from the promoter, and functions to repress transcription from the WT1 promoter in cell lines of non-renal origin. The 460-base pair silencer region is unusual in that it contains a full-length Alu repeat. We have also cloned an enhancer like-element located 1.3 kilobases upstream of the WT1 promoter.

Base Sequence↗
PubMed1995

GATA-1 transactivates the WT1 hematopoietic specific enhancer.

The Wilms' tumor gene, WT1, is believed to play a role in hematopoiesis as it is expressed in the spleen and in immature leukemias in addition to the developing genitourinary system. WT1 is down-regulated in differentiated leukemia cells both in vivo and in vitro and is up-regulated in fetal spleen and immature leukemia cells. The modulation of WT1 expression was examined in many cell types, and a hematopoietic-specific enhancer element has been identified. Here we describe the transcriptional response of this enhancer to hematopoietic-specific transcription factors. We found co-expression of WT1 and GATA-1 mRNA in K562 cells and in mouse spleen, suggesting potential interactions between these two transcription factors. We find that the activity of the 3' WT1 enhancer is positively correlated with the expression of GATA-1. Gel shift competition experiments and transactivation studies revealed that this functional activity is mediated via binding at a GATA-binding site in the WT1 enhancer. The transactivation of the WT1 enhancer by GATA-1 implies that GATA-1 plays a role in the regulation of WT1 during hematopoiesis.

Base Sequence↗
PubMed1995

Three novel aniridia mutations in the human PAX6 gene.

Aniridia (iris hypoplasia) is an autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the study reported here we describe PAX6 mutations in one sporadic and five familial cases with aniridia. Of the four different mutations identified, one was identical to a previously reported mutation (C-->T transition at codon 240), and three were novel: two in the glycine-rich region and one in the proline/serine/threonine-rich (PST) region. One PAX6 mutation found in the PST region was associated with cataracts in an aniridia family. Another splice mutation in the PST domain occurred in an aniridia patient with anosmia (inability to smell). The six new aniridia cases reported here have mutations predicted to generate incomplete PAX6 proteins. These results support the theory that human aniridia is caused by haploinsufficiency of PAX6.

Adult↗
PubMed1995

WT1 exon 1 deletion/insertion mutations in Wilms tumor patients, associated with di- and trinucleotide repeats and deletion hotspot consensus sequences.

The WT1 gene is known to play a role in at least some cases of Wilms tumor (WT). The first exon of the gene is highly GC rich and contains many short tandem di- and trinucleotide repeats, interrupted direct repeats, and CCTG (CAGG) motifs that have been identified as hotspots for DNA deletions. We have analyzed 80 WT patient samples for mutations in the first exon of WT1, either by SSCP analysis of the first 131 bp of the coding portion of WT1 exon 1 or by size analysis of a PCR product encompassing the coding region of exon 1 in addition to flanking noncoding regions. We report here the occurrence of somatic and germ-line deletion and insertion mutations in this portion of the gene in four WT patients. The mutations are flanked by short direct repeats, and the breakpoints are within 5 nt of a CCTG (CAGG) sequence. These data suggest that a distinctive mutational mechanism, previously unrecognized for this gene, is important for the generation of DNA mutations at the WT1 locus.

Base Sequence↗
PubMed1994

Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell type-specific enhancer and promiscuous promoter.

The Wilms' tumor gene, WT1, is expressed in few tissues, mainly the developing kidney, genitourinary system, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kidney and hematopoietic cell lines. We found three transcription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase II promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC-rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This finding suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identified a transcriptional enhancer located 3' of the WT1 gene > 50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested.

Base Sequence↗
PubMed1994

Testicular germ cell tumors of adults show deletions of chromosomal bands 11p13 and 11p15.5, but no abnormalities within the zinc-finger regions and exons 2 and 6 of the Wilms' tumor 1 gene.

We have studied the involvement of chromosomal bands 11p13 and 11p15.5 in 15 testicular seminomas (SE) and 18 testicular nonseminomatous germ cell tumors (NS). No allelic imbalances were found in 40% of the SE and 44% of the NS. Loss of heterozygosity (LOH) at 11p15.5 was seen in 21% of the SE and 47% of the NS; the corresponding frequencies for 11p13 were 47% and 44%. Both regions were deleted in 13% of the SE and 44% of the NS, indicating that all NS with a complete LOH of 11p13 also lost the 11p15.5 region. In one (out of two) SE and in five (out of eight) NS, this was due to at least two separate deletions. Loss of the whole p-arm was likely in one SE and two NS. No gross genomic changes of the Wilms' tumor 1 (WT1) tumor suppressor gene were found using a cDNA probe (WT33). Nor were aberrations found in the zinc-finger regions and exons 2 and 6 of this gene, using polymerase chain reaction amplification, single stranded DNA polymorphism analysis, and sequencing. We suggest that loss of genetic information from the short arm of chromosome 11, without affecting the WT1 gene in the regions studied, is relatively frequent but not crucial in the pathogenesis of testicular germ cell tumors of adults.

Adolescent↗
PubMed1994

Origin and biology of a testicular Wilms' tumor.

A pure triphasic testicular Wilms' tumor, without teratomatous elements, was studied using multiple techniques. Carcinoma in situ (CIS), the characteristic precursor of testicular germ cell tumors of adults (TGCTs), was found in the adjacent parenchyma. Flow cytometric analysis showed a single hypotriploid tumor stem line. Karyotyping of the tumor revealed some numerical and structural abnormalities, including an i(12p), the chromosomal marker of TGCTs. In situ hybridization supported the karyotypic findings, and showed a similar numerical distribution in CIS and the tumor. Molecular analysis of the tumor illustrated that all short arms of chromosome 12, including i(12p), were of maternal origin. No 12q deletions were detected. In spite of complete loss of the paternal 11p13 band, the zinc finger regions and exons 2 and 6 of the WT1 gene contained no aberrations. Therefore, this tumor suppressor gene is not inactivated due to aberrations in the studied regions. In addition, all four WT1 alternative transcripts were expressed in the tumor. No aberrations were found in chromosomal bands 11p15.5, 16q22.1, and 16q24. Both parental alleles of the human imprinted genes H19 and IGF2 were expressed in the tumor. This is the first report on the chromosomal and molecular characterization of an extrarenal Wilms' tumor. Its germ cell origin was unequivocally demonstrated.

Adult↗
PubMed1994

Paired box mutations in familial and sporadic aniridia predicts truncated aniridia proteins.

Aniridia, an autosomal dominant ocular disorder characterized by iris hypoplasia, results from mutations in the PAX6 gene, which encodes paired box and homeobox motifs. In this report we describe five new mutations in the paired box region of the human PAX6 gene that are associated with aniridia. The paired box mutations that we detected were in both familial (three) and sporadic (two) cases. All five mutations predict truncated PAX6 proteins. Our study indicates that early premature translational termination mutations in the PAX6 gene result in haploinsufficiency and generate the aniridia phenotype.

Amino Acid Sequence↗
PubMed1993

Wilms tumor genes.

Multiple 'WT' genes exist. The WT1 gene at chromosomal band 11p13 has been cloned and is known to be important in the etiology of at least some tumors by virtue of the identification of both germline and somatic mutations in WT patients. Genes at 11p15 and 16q are also involved, either as initiating or tumor progression events. An unlocalized familial predisposition gene is also known to be important etiologically. The identification of several genes that are involved in the etiology or progression of WT, the preferential loss of maternally derived alleles in tumor tissue, and the observed reduction to 11p homozygosity in normal tissue DNA from some patients, all strikingly indicate that a simple, one-locus-'two-hit' genetic model for WT is inadequate. The question is not if this model needs to be modified, but how it should be modified, or if it is even valid enough to be a starting point for understanding the genetics of Wilms tumor. To begin to address this, several questions can be asked. Do all Wilms tumors carry mutations at the WT1 locus? Do both alleles at the WT1 locus need to be inactivated or lost for tumorigenesis? Or, instead, do some WT1 mutations act dominantly? Do patients with bilateral disease carry germline mutations as originally hypothesized, or, as more recently suggested, is bilateral disease the result of early somatic mutations, genomic imprinting, or multifactorial inheritance? Must mutations at an 11p15 locus and/or 11p15 LOH accompany WT1 mutations, or do 11p13 and 11p15 mutations act independently of each other? Have tumors from familial WT cases (who do not carry germline WT1 mutations) sustained somatic mutations at the WT1 locus, the 11p15 locus or the 16q locus? Conversely, do tumors from sporadic WT patients carry somatic mutations at the non-11p familial predisposition gene? Will most tumors be found to carry mutations at the same one or two loci, but differ only with regard to whether the mutations are somatic or germline? Are effects of genomic imprinting layered over, so to speak, a framework of classically mendelian mutations, or in some cases is imprinting the mechanism by which genes are inactivated or their normal function modulated? Although not definitive, there are data that bear on some of these questions. Germline mutations have been observed in patients with bilateral tumors, but may not prove to be a universal feature of bilateral disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Genes, Wilms Tumor↗
PubMed1993

Transcriptional regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis.

The expression of a hematopoietic proteoglycan core protein (HpPG) gene is up-regulated during the early stages of myeloblast differentiation at a time point coinciding with the beginning of granule genesis (Stellrecht, C. M., Mars, W. M., Miwa, H., Beran, M., and Saunders, G. F. (1991) Differentiation 48, 127-135). The mechanism of this up-regulatory event was investigated by analyzing the expression and regulation of the HpPG gene during the differentiation of the pluripotent hematopoietic cell line, K562. The level of HpPG gene expression in these cells was up-regulated approximately 10-fold upon 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation, as measured by Northern blot analysis. The HpPG gene's expression remained relatively unchanged during hemin-induced erythroid differentiation, further demonstrating the specificity of this regulatory event for granule-producing cell lineages. The effect of TPA induction on HpPG gene expression was also assessed during the differentiation of the myeloid leukemia cell line, HL-60. The expression of the gene was down-regulated approximately 20-fold upon TPA-induced differentiation into macrophage-like cells. In contrast, only a minimal decrease in HpPG gene expression was detected in gamma-interferon-induced monocyte differentiation. No detectable changes in expression levels were seen in HL-60 cells differentiated into granulocytes with retinoic acid or dimethyl sulfoxide. Nuclear runoff analysis demonstrated that the regulation of the HpPG gene is under transcriptional control in both TPA-induced differentiation systems.

Aggrecans↗
PubMed1993

Genetic mosaicism in normal tissues of Wilms' tumour patients.

We describe the partial loss of heterozygosity (LOH) at chromosome 11p loci in normal tissues (normal kidney and/or blood) from four of 67 Wilms' tumour patients. Autologous tumour DNA showed complete loss of the same, maternally derived, alleles. These observations indicate that the normal tissues were mosaic for cells heterozygous and homozygous for 11p markers and that tumours subsequently developed from the homozygous cells that had undergone an 11p somatic recombination event. We suggest that LOH for 11p alleles is compatible with normal growth and differentiation and is significant pathologically only when accompanied by other genetic alterations.

Alleles↗