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.

Cell-free modulation of proinsulin synthesis.

In vivo, glucose preferentially stimulates proinsulin biosynthesis; at least part of this process is independent of new RNA synthesis and is accompanied by increases in the overall rate of polypeptide chain initiation. The cell-free translation of proinsulin messenger RNA is very sensitive to changes in the protein-synthesizing system. Proinsulin synthesis is preferentially inhibited by the addition of increasing quantities of polyadenylate-containing RNA from the fetal bovine pancreas or by the addition of the drug, aurintricarboxylic acid, which blocks polypeptide chain initiation. These results suggest that proinsulin messenger RNA completes less efficiently for rate controlling initiation factors. We propose that glucose stimulates proinsulin biosynthesis by allowing the less competitive proinsulin messenger RNA to be translated more efficiently.

The human placental lactogen genes: structure, function, evolution and transcriptional regulation.

hPL is a member of an evolutionarily related gene family including hGH and hPRL. Expression of hPL is limited to the placenta but its physiological actions are far reaching. hPL has a direct somatotropic effect on fetal tissues, it alters maternal carbohydrate and lipid metabolism to provide for fetal nutrient requirements, and aids in stimulation of mammary cell proliferation. Two hPL genes (hPL3 and hPL4) encoding identical proteins are responsible for the production of up to 1-3 g PL hormone/day. Recent studies have characterized the regulatory controls of hPL expression. At the post transcriptional level, RNA stability may contribute to variable levels of hPL3 vs. hPL4 production. In addition, non-tissue-specific protein-promoter interactions involving the Sp1 transcription factor are necessary for hPL transcription initiation. A transcriptional enhancer located 3' to the hPL3 gene is responsible for the placenta-specific expression of this gene, while an additional enhancer may be located 3' to the hPl4 gene. The hPL enhancer is bound by multiple proteins including at least one placental specific protein that interacts with a TEF-1 motif. Therefore, enhancer-protein interactions most likely play a large part in the high levels of placenta-specific hPL expression.

Activation of the human PAX6 gene through the exon 1 enhancer by transcription factors SEF and Sp1.

PAX6 is a transcription factor that plays a major role in ocular morphogenesis. PAX6 is expressed in the eye, central nervous system and pancreas. Two alternative promoters, P0 and P1, which are differentially regulated during development, drive PAX6 transcription. We identified a 57 bp cis-regulatory element in exon 1 of the human PAX6 gene exon 1 enhancer (EIE). EIE enhances P1-driven PAX6 expression. Three regions in E1E (E1E-1, E1E-2 and E1E-3) have sequence similarities with binding sites of transcription factors ARP-1, Isl-1 and SEF, respectively. As shown by electrophoretic mobility shift assays, E1E-3, but not E1E-1 or E1E-2, bound to proteins in nuclear extracts of human glioma cells and transcription factor SEF bound to E1E-3. As shown by transient transfection experiments, deletion or site-specific mutations in E1E-3 dramatically decreased P1 promoter activity. Mutations in E1E-2, however, did not affect function of the P1 promoter. Co-transfection of SEF and PAX6 promoter-reporter constructs showed that SEF up-regulates PAX6 gene expression through the P1 promoter. Two Sp1 sites in the E1E region were also shown to be important by transient co-transfection assays. Data from immunoprecipitation and transient transfection assays demonstrated that SEF and Sp1 interacted in vitro and may act together in vivo to regulate PAX6 expression.

RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.

Effect of bleomycin on the synthesis and function of RNA.

Bleomycin inhibits cellular RNA synthesis and the inhibition is nonspecific. The ratio of polyadenylate- [poly(A)] containing RNA to non-poly(A)-containing RNA in the drug-treated human lymphocytic cells, line Wil2, was the same as that in untreated cells. Poly(A) RNA isolated from untreated cells was used as a template for reverse transcriptase to synthesize complementary DNA, which was then used as a probe to assay the sequence diversity of poly(A)RNA's from treated and untreated cells. It was found that essentially all of the poly(A) RNA's in the untreated cells were also present in the treated cells. The effect of bleomycin on the biological activity of messenger RNA (mRNA) was tested with globin mRNA in a wheat germ embryo translation system. Although bleomycin inhibited protein synthesis at high concentrations, the inhibition was not due to a modification of mRNA. This was evidenced by the fact that no decrease in the ability of mRNA to function in the test system was found when globin mRNA was pretreated with high concentrations of bleomycin followed by removal of the drug.

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.

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)

Expression of the Wilms' tumor gene (WT1) in human leukemias.

Leukemic cells from seventy patients with various types of human leukemias were examined for expression of the WT1 gene, the Wilms' tumor gene located at chromosome 11p13. WT1 was expressed in 7 of 16 cases of acute lymphoblastic leukemia, 15 of 22 with acute myelogenous leukemia and 8 of 10 in blast crisis of chronic myelogenous leukemia. No detectable WT1 RNA was found in chronic leukemias, including chronic lymphocytic leukemia, plasma cell leukemia, hairy cell leukemia and chronic myelogenous leukemia in chronic phase. The expression pattern of WT1 in these human leukemia samples indicates the involvement of this gene in the early stage of hematological cell differentiation.

HLA-associated invariant chain gene expression in human B cell neoplasia.

The expression of the gene encoding the HLA-DR associated invariant chain (In-gene) in human B lymphocytes was analyzed by determining the level of invariant chain, mRNA in peripheral blood and bone marrow cells of several patients affected by hematological malignancies. In B cell neoplasms representative of different stages of B lymphocyte differentiation, In-gene activation was an early event that may occur in pre-B cells before immunoglobulin gene transcripts are detectable. The highest level of invariant chain mRNA were observed at an intermediate maturation stage corresponding to sIg, Ia, and B1 positive peripheral blood lymphocytes. At the terminal stage of B lymphocyte differentiation, the In-gene was turned off. In leukemic cell populations, the pattern of temporal activation of the In-gene corresponded to the pattern of activation of the genes encoding the HLA-DR alpha and beta chain.

WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia.

The Wilms' tumor protein, WT1, represses transcription from several growth factor genes. WT1 transcription is regulated in erythroid and myeloid lineages by the transcription factor GATA-1. Using a sensitive, isotopic duplex RT-PCR procedure amplifying WT1 or GATA-1 together with beta-actin as the internal control in a single reaction mix, we quantitated the expression of WT1 and GATA-1 mRNA of 16 patients with myelodysplastic syndrome (MDS), 56 with acute myeloid leukemia (AML) and 22 with acute lymphoblastic leukemia (ALL). K562 was used as reference positive control for this cell line expresses both WT1 and GATA-1. Among MDS patients, increased WT1 expression was found in refractory anemia with excess blast (RAEB) and RAEB in transformation (RAEB-T) subtypes compared to the normal controls, whereas WT1 expression in refractory anemia (RA) was not different from the normal control level. All of AML cases of subtypes M0, M1, M2 and M3 expressed WT1 more than three times the normal WT1 level. Subtypes M4 to M7 showed significantly lower WT1 levels than M1 to M3 and AML cases with CD14+ expressed less WT1 than CD14-. Higher than normal WT1 levels were also expressed in cases of ALL.

Localization of an abundant myeloid mRNA to individual leukocytes in mixed cell populations.

Differential screening of cDNA libraries with radiolabeled RNAs isolated from various sources provides a convenient way to identify cDNA clones representing RNAs that are more (or less) abundant in selected tissues. This strategy was previously used to isolate cDNA clones representing poly(A+)RNAs (mRNA) that are abundant in leukocytes from chronic myelogenous leukemia (CML) patients. One limitation with the initial experiments was that the RNAs were isolated from heterogenous cell populations and it was impossible to distinguish whether all of the cells were producing the abundant mRNAs or if a subset of the cell population was responsible for the majority of the RNA species. To resolve this important issue, we have directly hybridized radiolabeled cDNAs to the cellular RNAs of intact, morphologically distinguishable, primary hematopoietic cells. In the present study clone pC-A3, which represents an mRNA species that is abundant in the chronic phase of CML, was used to examine three samples from normal bone marrow, one sample from normal peripheral blood, and four samples from peripheral blood of leukemic patients (one Ph1 + AL, two CML in compensated phase, and one CML in accelerated phase). The results show that while C-A3 gene expression is detectable in cells from the granulocytic, monocytic, and lymphoid lineage, its relative abundance peaks at the level of the neutrophilic myelocytes and promyelocytes. Earlier myeloid precursors like myeloblasts or mature neutrophilic granulocytes show less labeling. Further, all maturation stages of eosinophils (Eos) and basophil (Baso) are highly labeled. This finding reinforces recent evidence that Eos and Baso share a common progenitor and suggests that these two cell types may have a stronger role than previously noted in the prominent myeloproliferative response that is characteristic of CML.

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.

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.

DNA sequences involved in the transcriptional activation of a human placental lactogen gene.

To identify regulatory elements in the promoter of a human placental lactogen gene (hPL3) that are important for its transcriptional activation, sequences 5' to the start of transcription were linked to the reporter gene chloramphenicol acetyltransferase (CAT) and transiently transfected into JEG-3 cells, a human placental choriocarcinoma cell line. In the presence of the hPL3 enhancer, deletion of the promoter sequence between -142 and -129 basepairs resulted in an 8-fold decrease in CAT activity. Similar results were seen with the SV40 enhancer and the hPL3 promoter in HepG2 liver cells. Nuclear proteins from HepG2, HeLa, and JEG-3 cells formed specific binding complexes with this region of the hPL3 promoter by a gel mobility shift assay, indicating that the DNA-binding protein was not tissue specific. The -142 to -129 basepair region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. An oligonucleotide containing Sp1-binding sites specifically competes for proteins binding the hPL3 promoter, and the methylation interference pattern is similar to that for an Sp1-binding site. This suggests that the hPL3 promoter binds Sp1- or an Sp1-like trans-acting factor, and this binding site is important for transcriptional regulation by the hPL3 enhancer in PL-producing cells.

Structure and evolution of somatostatin genes.

A bovine pancreatic preprosomatostatin cDNA clone has been isolated and sequenced. Although it encodes a predicted 116 amino acid preprosomatostatin that is very similar in primary structure to those deduced from other mammalian preprosomatostatin cDNAs, there are some differences in amino acid composition. Hybridization of this clone to Northern blots of fetal bovine pancreatic poly(A+) RNA reveals a mRNA of 700 nucleotides. Evolution of the preprosomatostatin genes was studied by statistical analysis of anglerfish, catfish, bovine, rat, and human cDNA sequences. The results suggest that the two somatostatin genes present in both anglerfish and catfish were the result of a gene duplication event in a common ancestor of anglerfish and catfish.

Nucleotide sequence of a transcriptional enhancer located 2.2 kb 3' of a human placental lactogen-encoding gene.

The nucleotide sequence of the human placental lactogen-encoding gene enhancer was determined. This tissue-specific enhancer is contained in a region flanked by a 284-bp Alu repeat.

A novel Pax-6 binding site in rodent B1 repetitive elements: coevolution between developmental regulation and repeated elements?

Pax-6 encodes a transcription factor that is important in the development of eye and CNS. Identification of Pax-6 target genes is crucial for understanding the gene regulatory network in these developmental processes. Using an in-vitro approach of cyclic amplification of the protein binding sequences (CAPBS), we isolated a PAX6 binding sequence from a human single-copy (sc) DNA library. Characterization of this PAX6 binding sequence revealed a 15bp region (hGCalpha1BLs5) that is sufficient for PAX6 specific binding. From a homology search in the GenBank, we found that an hGCalpha1BLs5-like Pax-6 binding site exists in 21 genes (16 from rodent), 15 of which were shown to be able to bind Pax-6 in vitro. Interestingly, some of these sites occur in B1 repetitive elements. Although hGCalpha1BLs5 is highly similar to a region in B1 repetitive elements, PAX6 does not bind to the consensus sequence in B1. However, a single-step mutation in some B1 elements can lead to a gain of function for PAX6 binding. This experimental evidence and phylogenetic analysis raise an interesting speculation for the coevolution between PAX6 regulation and repeat elements. Since a (Pax-6-binding) null B1 element can be re-activated by even a single-step mutation, it has the potential to recruit gene targets for Pax-6 if it is inserted into the regulatory region, and therefore may play a role for evolutionary modification of Pax-6 regulation.

Isolation and characterization of preproinsulin mRNA from fetal bovine pancreatic islets.

Total polyadenylated RNA prepared from isolated islets of fetal bovine pancreas was physically characterized. Incubation of this poly A+ RNA with a cell-free protein-synthesizing system derived from wheat germ results in the synthesis of insulin-immunoreactive polypeptide identical in size to that described earlier, 11 200 daltons (Lomedico et al. (1977) J. Biol. Chem. 252, 7971--7978). This material comprised approximately 22% of the total 3H-labeled translation products. Compared to poly A+ RNA from the total pancreas, we conclude that islet mRNA is enriched in proinsulin mRNA.

Differentially spliced exon 5 of the Wilms' tumor gene WT1 modifies gene function.

The Wilms' tumor gene WT1 encodes a zinc-finger transcription factor that functions as a tumor suppressor gene and repress transcription of a number of growth factors and proto-oncogenes. In the developing kidney, WT1 expression peaks at the onset of the mesenchyme-to-epithelium transition and is required for epithelial differentiation. WT1 mRNA undergoes alternative splicing at two sites, resulting in four mRNA species and proteins expressed in constant ratios. The first alternative splice results in the presence or absence of exon 5, which is 51 nucleotides long and encodes 17 amino acids between the amino-terminal, proline-rich transcriptional repression domain and the carboxy-terminal DNA-binding zinc-fingers. We used cell-proliferation assays to determine the effect of exon 5 on WT1 function. Isoforms of WT1 without exon S repressed cell growth. WT1 isoforms with exon 5 slowed cell growth to a lesser extent but resulted in altered cellular morphology. These results provide evidence that WT1 splice isoforms differentially regulate cell proliferation and initiate the mesenchyme-to-epithelium transition during metanephric development.