Connexin 26 enhances the bystander effect in HSVtk/GCV gene therapy for human bladder cancer by adenovirus/PLL/DNA gene delivery.

Herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) gene therapy has been used for the treatment of a variety of cancers. Its efficacy is enhanced by the bystander effect that helps overcome the delivery problems commonly observed in current gene therapy. Connexins encode proteins that produce gap junctions, which enable intercellular communication and the bystander effect. We previously demonstrated that decreased Cx 26 expression and loss of gap junctional intercellular communication were associated with human bladder cancer. To investigate the efficacy of the bystander effect in HSVtk/GCV gene therapy, the Cx 26 gene was introduced into UM-UC-3 and UM-UC-14 bladder cancer cell lines by an adenovirus poly-L-lysine conjugate using a multigenic expression plasmid that expressed both the HSVtk and Cx 26 genes. We found significantly increased cytotoxicity in HSVtk/GCV gene therapy after introduction of the HSVtk and Cx 26 genes together compared with the cytotoxicity seen after introduction of the HSVtk gene and LacZ genes in vitro and in vivo. Cytotoxicity correlated with Cx 26 expression and the induction of functional gap junctions. This study indicates that combination gene therapy with co-expression of the HSVtk and Cx 26 genes potentiates HSVtk/GCV gene therapy through the bystander effect.

Transcriptional regulation of the androgen signaling pathway by the Wilms' tumor suppressor gene WT1.

The androgen-signaling pathway plays a critical role in prostate cancer development and progression. We have recently demonstrated that the Wilms' tumor suppressor gene product, WT1, binds to multiple sites in the androgen receptor (AR) promoter and transcriptionally represses the AR gene promoter in vitro. We asked whether WT1 repression of the endogenous AR gene interferes in the androgen signal transduction cascade and modifies AR target gene expression. We analyzed the effect of WT1 (-/-) overexpression on an AR target gene reporter construct that contains the luciferase gene, the ElB TATA box, and two copies of the androgen-response element (ARE), the dimeric AR binding site. Luciferase activity was determined in 293 kidney and TM4 Sertoli cells, two nontumorigenic cell lines that express both AR and WT1. Cells were cotransfected by lipofectamine in the presence or absence of the synthetic androgen R1881. Results showed that overexpression of WT1 downregulates ARE-reporter gene transcription in both cell lines tested. The inhibitory effect of WT1 on the AR target gene construct was dose-dependent and androgen-independent in 293 cells, whereas in TM4 cells it was androgen-dependent. Additionally, a zinc-finger mutant WT1 (-/-) expression construct, R394W, failed to decrease luciferase activity, suggesting that WT1 downregulates the ARE-reporter gene construct activity by directly repressing the endogenous AR gene promoter. Furthermore, we analyzed the expression of WT1 and AR mRNA in several prostate cancer cell lines in order to understand the role WT1 may play in prostate cancer development and progression. Gel analysis of cDNA amplified by RT-PCR of AR and WT1 RNA from prostate cancer and non-prostatic cell lines showed that LNCaP and MDAPCa2b, two metastatic prostate cancer cell lines which are androgen-sensitive, expressed AR but not WT1. Du145 and PC3, two cell lines from advanced metastatic prostate cancer, which are characterized as androgen-independent and -insensitive, did not express AR but expressed a high level of WT1. Two non-prostatic cell lines, T47D and 293, weakly co-expressed AR and WT1. This inverse relationship between AR and WT1 expression in prostate cancer cell lines, together with WT1 repression of the AR promoter, suggest a role for WT1 in the androgen signaling pathway and in prostate cancer development and progression.

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.

Transactivation of an intronic hematopoietic-specific enhancer of the human Wilms' tumor 1 gene by GATA-1 and c-Myb.

The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor which is expressed in a tissue-specific manner. Our studies indicate that in addition to the promoter, other regulatory elements are required for tissue-specific expression of this gene. A 258-base pair hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8-10-fold in K562 and HL60 cells. Sequence analysis revealed both a GATA and a c-Myb motif in the enhancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that the GATA-1 protein in K562 nuclear extracts binds to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 expression vector showed that GATA-1 transactivated this enhancer, increasing the CAT reporter activity 10-15-fold. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has also been mapped in the 258-base pair enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo.

PAX 8 regulates human WT1 transcription through a novel DNA binding site.

The Wilms' tumor gene (WT1) is an essential gene for kidney and gonadal development, although how WT1 expression is induced in these tissues is not known. One kidney transcription factor likely to play a role in this regulation is PAX 8. The co-expression of WT1 and PAX 8 during kidney development and in Wilms' tumors with an epithelium predominant histology suggested a possible interaction, and indeed, we identified potential core PAX-binding sites in the WT1 promoter. Endogenous PAX 8 plays an important role in the activation of the WT1 promoter, since promoter activity is much stronger in cells with PAX 8 than without. Using binding assays, we searched for evidence of PAX 8-DNA interactions throughout the 652-base pair human WT1 promoter and found only one functional PAX 8 site with DNA binding activity, located 250 base pairs 5' of the minimal promoter. The responsiveness of the PAX 8 site was confirmed by assessing its ability to function as an enhancer significantly activating the minimal promoter in a position- and orientation-independent manner. Using transfection assays, we demonstrated that either endogenous or exogenously added PAX 8 activated the WT1 promoter and that this promoter up-regulation depended upon the presence of an intact PAX 8-binding site. In contrast, the previously reported core PAX 8-binding sites identified by computer analysis of the WT1 promoter failed to specifically bind in vitro translated PAX 8 protein or activate the minimal promoter. Thus, we identified a novel functional binding site for the transcription factor PAX 8, suggesting that part of its role in kidney development may be as a modulator of WT1 expression in the kidney.

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.

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.

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.

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.

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.

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.

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.

Deletion/frameshift mutation in the alpha 1-antitrypsin null allele, PI*QObolton.

The most common deficiency allele of the protease inhibitor (PI) alpha 1-antitrypsin (alpha 1AT) is PI*Z. Other rare deficiency alleles of alpha 1AT are of two types: those producing low but detectable amounts of alpha 1AT (less than 20% of normal serum concentrations), and null alleles producing less than 1% of normal alpha 1AT and therefore not detectable by routine quantitative methods. We have previously used DNA polymorphisms and family data to determine heterozygosity in an individual producing low levels of serum alpha 1AT (12% of normal) of PI type Mmalton. By DNA analysis we observed the typical haplotype associated with PI*Mmalton and a unique null haplotype associated with the allele PI*QObolton. The QObolton allele produces no detectable serum alpha 1AT. We have cloned and sequenced the QObolton allele from a phage genomic library. Deletion of a single cytosine residue near the active site of alpha 1 AT in exon V results in a frameshift causing an in-frame stop codon downstream of the deletion. This stop codon leads to premature termination of protein translation at amino acid 373, resulting in a truncated protein. The truncated protein is predicted to have an altered carboxy terminus (amino acids 363-372) and will lack structurally important amino acids.

In-frame single codon deletion in the Mmalton deficiency allele of alpha 1-antitrypsin.

A deficiency of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT) is usually a consequence of the PI*Z allele. Mmalton is another deficiency allele which, like Z alpha 1AT, is associated with hepatocyte inclusions and impaired secretion. We report here the sequence of the PI Mmalton allele, which contains a 3-bp deletion coding for one of two adjacent phenylalanine residues (amino acid 51 or 52 of the mature protein). Using oligonucleotide hybridization of polymerase chain reaction-amplified DNA, we have demonstrated cosegregation of the PI Mmalton protein and the 3-bp deletion in the family in which this allele was originally described and in three other, unrelated kindreds. This deletion is found exclusively in PI Mmalton alleles and not in the normal M2 alleles from which, to judge on the basis of haplotype data, the Mmalton mutation must have been derived. In polyacrylamide isoelectric focusing (PIEF) gels, the isoelectric point of Mmalton is only slightly more cathodal than M2, a finding consistent with the loss of a single uncharged amino acid. To judge on the basis of X-ray crystallography data for the normal alpha 1AT protein, the deletion of aa 51/52 would shorten one strand of the beta sheet, B6, apparently preventing normal processing and secretion.

Association of increased platelet-derived growth factor secretion and retroviral expression in transformed mouse and human cells.

The activation of platelet-derived growth factor (PDGF) production by transformed cells is often observed but not well understood. We have examined cell lines that showed "spontaneous" increases in PDGF secretion, i.e., in which the increase was not in response to intentional intervention. In one case the increase was associated with an obvious change in morphology and mitogen requirements accompanying spontaneous transformation of Swiss 3T3 cells. In the other case the increase occurred during growth of a human tumor cell in a nude mouse and was not associated with an alteration in the morphology or growth properties of the cells. Rates of PDGF secretion did not correlate with specific changes in the pattern of expression of PDGF mRNA. In the human tumor system PDGF A- and B-chain transcripts were present at similar levels before and after transplantation in the nude mouse. In the 3T3 cell system, B-chain transcripts were detected only after transformation, and there was no change in the low basal expression of A-chain. A change which did consistently correlate with the increased secretion of PDGF was that both the spontaneously transformed murine cells and the transplanted human cells expressed murine leukemia virus transcripts and synthesized retroviral envelope glycoproteins, while their original counterparts did not.

Production of platelet-derived growth factor by cultured Wilms' tumor cells and fetal kidney cells.

We have cultured human Wilms' tumors (nephroblastomas) in vitro and examined their growth properties, tumorigenicity in nude mice, karyotypes, and production of a platelet-derived growth factor (PDGF)-like activity (PDGFc). The cultured Wilms' tumor cells do not exhibit transformed growth properties, senesce after 15-25 passages, and are not tumorigenic in nude mice. Two of the three Wilms' tumors examined had karyotypic abnormalities but none contained a deleted 11p region. The Wilms' tumor cells and control human fetal kidney cells produce significant amounts of PDGFc and express few PDGF receptors on the cell surface. Analysis of Wilms' tumor cell RNA using probes specific for PDGF A-chain and B-chain (c-sis) detects A-chain but not B-chain transcripts in the tumor cells.

Isolation and preliminary characterization of mutants of western equine encephalomyelitis virus with altered virulence in chickens.

A small plaque (sp) and four temperature-sensitive (ts) mutants of western equine encephalomyelitis virus (WEEV) were obtained from a highly virulent field isolate (BFS-1703) after mutagenesis with 5-azacytidine or nitrosoguanidine or following undiluted passage in mosquito cell culture. Three ts mutants (158, 214, and 260) expressed stable ts phenotypes as determined both by six-hour leak yield and efficiency of plaquing (EOP) analyses. Progeny of ts mutant 214 grown at the nonpermissive temperature lacked hemagglutinin activity. The sp mutant (148) was as highly virulent as the wild-type virus (WTV) for newly hatched chickens (infected when 12 hours old), whereas the ts mutants were attenuated in their virulence for this host. Attentuation was correlated with lower viremia titers in 24-hour postinfection blood samples. The WTV and those mutants able to infect 21-day-old chickens produced nonlethal infections but the growth of ts 158 was apparently restricted in this high-temperature nonpermissive host. All ts mutants except ts 158 lost their ts phenotype after growth for 24 hours in newly hatched chickens and in duck embryonic cell culture (DECC) maintained at 42 degrees C.