Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumor pathogenesis.

Wilms' tumor (WT), one of the most common pediatric solid cancers, arises in the developing kidney as a result of genetic and epigenetic changes that lead to the abnormal proliferation and differentiation of the metanephric blastema. As activation of signal transducers and activators of transcription (STATs) plays an important role in the maintenance/growth and differentiation of the metanephric blastema, and constitutively activated STATs facilitate neoplastic behaviors of a variety of cancers, we hypothesized that dysregulation of STAT signaling may also contribute to WT pathogenesis. Accordingly, we evaluated STAT phosphorylation patterns in tumors and found that STAT1 was constitutively phosphorylated on serine 727 (S727) in 19 of 21 primary WT samples and two WT cell lines. An inactivating mutation of S727 to alanine reduced colony formation of WT cells in soft agar by more than 80% and induced apoptosis under conditions of growth stress. S727-phosphorylated STAT1 provided apoptotic resistance for WT cells via upregulation of expression of the heat-shock protein (HSP)27 and antiapoptotic protein myeloid cell leukemia (MCL)-1. The kinase responsible for STAT1 S727 phosphorylation in WT cells was identified based upon the use of selective inhibitors as protein kinase CK2, not p38, MAP-kinase kinase (MEK)1/2, phosphatidylinositol 3'-kinase, protein kinase C or Ca/calmodulin-dependent protein kinase II (CaMKII). The inhibition of CK2 blocked the anchorage-independent growth of WT cells and induced apoptosis under conditions of growth stress. Our findings suggest that serine-phosphorylated STAT1, as a downstream target of protein kinase CK2, plays a critical role in the pathogenesis of WT and possibly other neoplasms with similar STAT1 phosphorylation patterns.

K-ras activation in premalignant and malignant epithelial lesions of the human uterus.

We previously reported (Cancer Res., 50:6139-6145, 1990) a significant frequency of activating point mutations in codon 12 of the K-ras oncogene in endometrial adenocarcinomas of the uterine corpus (series 1). To further define the role of ras activation in the development of endometrial adenocarcinoma, we surveyed cystic, adenomatous, and atypical hyperplasias of uterine endometrium and additional cases of endometrial and cervical carcinoma (series 2) for the presence of activating mutations in cellular protooncogenes of the ras family. Polymerase chain reaction was performed from deparaffinized sections of formalin-fixed paraffin-embedded tissue. We screened for point mutations in codons 12, 13, and 61 of the K-, H-, and N-ras genes by dot blot hybridization analysis with mutation-specific oligomers. Mutations in K-ras were also confirmed by direct genomic DNA sequencing. Of 19 endometrial adenocarcinomas in series 2, point mutations in ras genes were found in 7 tumors. Six contained single-base substitutions, five in codon 12 of K-ras and one in codon 12 of N-ras. The seventh tumor contained two different point mutations in codon 12 of K-ras. In one endometrial adenocarcinoma, tumor cells with point mutations in K-ras were predominantly localized to a portion that had a more aggressive histological pattern. In endometrial hyperplasia, K-ras mutations, one in codon 12 and one in codon 13, were found in 2 of 16 hyperplasias histologically classified as atypical and clinically considered premalignant. None of 6 adenomatous hyperplasias and none of 12 cystic hyperplasias, the latter of which is considered clinically benign, contained any detectable ras mutations. No mutations in H-ras were detected in either carcinomas or hyperplastic tissue.

Epidermal growth factor and transforming growth factor-alpha-associated overexpression of cyclin D1, Cdk4, and c-Myc during hepatocarcinogenesis in Helicobacter hepaticus-infected A/JCr mice.

Helicobacter hepaticus is a new bacterial species that is homologous to Helicobacter pylori, a human gastric carcinogen. H. hepaticus causes chronic active hepatitis, with progression to hepatocellular tumors. We hypothesized that chronic up-regulation of epidermal growth factor (EGF), transforming growth factor-alpha, and nuclear oncogenes (cyclin D1 and c-Myc), all known to transform by overexpression, might contribute to tumorigenesis. Livers from mice that were 6-18 months old were analyzed, including nonneoplastic and preneoplastic tissues and tumors, along with age-matched controls, by immunohistochemistry and immunoblotting. EGF and transforming growth factor-alpha were increased at the earliest stage, with a further increase in EGF in tumors. Cyclin D1, cyclin-dependent kinase 4, and c-Myc were strongly increased in all infected livers, with even greater increases in tumors. An increase in cyclin D1/cyclin-dependent kinase 4 complex was also demonstrated in tumors, and its functionality was confirmed by an increase in the hyperphosphorylated:hypophosphorylated retinoblastoma protein ratio. Our findings suggest a possible cooperation of growth factors, cell cycle proteins, and transcription factors during the development of H. hepaticus-associated liver tumors and may have relevance to human cancers associated with bacterial, viral, or parasitic infections.

K-ras activation in neoplasms of the human female reproductive tract.

The role of cellular oncogenes in the development of epithelial tumors of the human female reproductive tract has not previously been extensively studied. DNAs isolated from ten human uterine, 13 ovarian, and four cervical neoplasms and from three cell lines derived from endometrial adenocarcinoma were investigated by dot blot hybridization after polymerase chain reaction amplification of ras gene sequences and in some cases by NIH 3T3 transfection. Transforming activity was found in two of nine endometrial adenocarcinomas, but none of seven ovarian carcinomas and none of four cervical carcinomas showed transforming activity. K-ras sequences with a GGT----GAT mutation in codon 12 were demonstrated in both transformants derived from endometrial carcinoma. K-ras codon 12 mutations were similarly detected in six of 13 endometrial carcinomas (one GAT and GCT, one GTT and GCT, two GAT, two GTT) and two of 13 ovarian tumors (GAT and GCT, GAT), both mucinous adenocarcinomas. Point mutation of K-ras in codon 12 is thus comparably frequent in uterine endometrial carcinomas and in colorectal carcinomas and may have similar significance as an event that contributes to progression of these tumors. Cervical carcinomas and ovarian tumors in general, with the possible exception of mucinous adenocarcinoma of the ovary, do not appear to have this characteristic.

Infrequent activation of K-ras, H-ras, and other oncogenes in hepatocellular neoplasms initiated by methyl(acetoxymethyl)nitrosamine, a methylating agent, and promoted by phenobarbital in F344 rats.

Fischer 344/Ncr rats of both sexes were subjected to partial hepatectomy and then initiated 21-24 h later by a single injection of methyl(acetoxymethyl)nitrosamine at 0.1 mmol/kg body weight via the portal vein. Beginning 3 weeks later, development of hepatocellular neoplasms in initiated rats was promoted by feeding 0.05% phenobarbital (PB) in the diet. Not only intrahepatic lesions but also a variety of extrahepatic tumors were induced. High-molecular-weight DNAs were prepared from 67 samples of grossly normal liver containing multiple preneoplastic foci/areas of microscopic dimensions, 137 hepatocellular adenomas (nodules), 93 hepatocellular carcinomas (HCC), 10 cholangiomas, and 25 extrahepatic tumors in 95 rats and tested for transforming activity in the NIH 3T3 transfection assay. DNA preparations from 7 of 93 HCCs, 2 of 10 cholangiomas, 2 of 137 nodules, 1 histiocytic sarcoma, and 1 thyroid carcinoma were positive in the transfection assay. Southern blot analysis showed that NIH 3T3 transformants induced by DNA from 5 HCCs, 1 hepatocellular adenoma, 1 cholangioma, 1 histiocytic sarcoma, and 1 thyroid carcinoma contained an activated K-ras gene of rat origin. Rat-derived H-ras was identified in transformants from 2 additional HCCs and rat c-raf from 1 hepatocellular adenoma. The transforming gene from one cholangioma showed no sequence homology to the ras genes, neu, or c-raf. Immunoprecipitation analysis of ras Mr 21,000 protein in 11 transformants indicated that, based upon protein electrophoretic mobilities, activation of the ras genes consistently resulted from mutations in codon 12 of these genes. Selective oligonucleotide analysis revealed that a G----A transition in the second base of codon 12 of K-ras was present in the 9 K-ras-positive transformants and also in DNAs prepared from the original tumors. In contrast, oligonucleotide hybridization experiments with DNAs from 35 hepatocellular tumors that were negative in transfection assays revealed the presence of mutant K-ras in 1 of 15 HCCs; no mutation could be detected in 20 transfection-negative adenomas. The infrequency of detection of a specific oncogene, more frequent detection of oncogenes in malignant tumors, and failure to observe activated oncogenes in preneoplastic lesions suggest that activation of ras oncogenes may occur as a late and infrequent event in the evolution of some rat hepatocellular neoplasms and that mutation of a specific ras locus is not an obligatory early event in the genesis of these neoplasms.

GGT to GTT transversions in codon 12 of the K-ras oncogene in rat renal sarcomas induced with nickel subsulfide or nickel subsulfide/iron are consistent with oxidative damage to DNA.

Nickel is a toxic, mutagenic, and carcinogenic metal of significant occupational and environmental concern. Although several cellular targets of nickel have been identified, considerable evidence suggests that it can act indirectly upon DNA by inducing the formation of oxidized purines or pyrimidines that constitute promutagenic lesions. In this study, we examined nickel subsulfide (Ni3S2)- or Ni3S2/iron-induced renal sarcomas in F344 rats for the presence of transforming mutations in the K-ras oncogene. Selective oligonucleotide hybridization analysis of K-ras gene sequences amplified by polymerase chain reaction revealed that 1 of 12 primary tumors induced with Ni3S2 and 7 of 9 primary tumors induced with Ni3S2/iron contained exclusively GGT to GTT activating mutations in codon 12. These mutations are consistent with the known ability of nickel, in the presence of an oxidizing agent, to catalyze formation of 8-hydroxydeoxyguanosine, which in turn promotes misincorporation of dATP opposite the oxidized guanine residue. The presence of GGT to GTT transversions was confirmed by direct sequencing of the polymerase chain reaction products. Sequencing also revealed that there were no transforming mutations in codons 13 or 59-61. Additionally, a direct correlation between shortened tumor latency and the presence of activating ras mutations was noted. These results show that, in rat kidney, Ni3S2 can induce transforming mutations that are consistent with the ability of nickel to produce oxidative lesions and that iron, which exacerbates the extent of cellular oxidative damage, can enhance the frequency of these transforming mutations.

Comparative immunohistochemical reactivity of monoclonal and polyclonal antibodies to H-ras p21 in normal and neoplastic tissues of rodents and humans.

Five mouse monoclonal antibodies (mES 2, 4, 8, 13 and 20) produced against bacterially expressed BALB ras p21 and five other monoclonal or polyclonal antibodies (Cetus rabbit or mouse pan p21, RAP-5, Triton Ha-ras, Y13-259) to H-ras p21 were used for comparative immunohistochemical detection of H-ras p21 by the avidin biotin peroxidase-complex technique in selected normal fixed tissues and tumors from rats, mice, and humans. Nine of these antibodies were immunoreactive with cell membranes and cytoplasm of harvey sarcoma virus-induced sarcoma cells, but usually only with Bouin's fixed tumors. A few normal tissues were immunoreactive with some of the antibodies except for many immunoreactive tissues found with mES 13. Although mES 13 stained sarcoma cells on the cell membrane, a prominent granular staining, which appeared to be mitochondrial or lysosomal, was seen in many normal and neoplastic rodent tissues and in normal human colon, colon polyps, and carcinomas. Interpretation of positive immunoreactivity with any antibody was sometimes difficult due to nonspecific (background) staining. The cellular pattern of specific reactivity (membrane or granular) and inhibition of immunoreactivity by absorption of the antisera with H-ras p21 was therefore important. Western blots with BALB transforming (Lys 12) p21 expressed in E. coli revealed reactivity of all antibodies except for RAP-5. In addition, immunoblots of solubilized proteins from the EJ cell line with RAP-5 indicated reactivity of this monoclonal antibody with proteins of several different molecular weights. RAP-5 also never specifically immunoreacted with cell membranes of normal or malignant cells including EJ cells. Interpretation of these findings in comparison with those in published reports of H-ras p21 localization in fixed tissue sections is discussed, including the importance of fixative, specific antibody and controls.

Activation of the K-ras gene by insertion mutations in chemically induced rat renal mesenchymal tumors.

Previously we reported the detection of transforming K-ras sequences in methyl(methoxymethyl)nitrosamine (DMN-OMe)-induced rat renal mesenchymal tumors by NIH3T3 transfection assays. Subsequent analysis by selective oligonucleotide hybridization revealed a variety of activating point mutations in codon 12 of K-ras in most of these tumors and in their NIH3T3 transformants, but in some, point mutations could not be detected by this technique. In the current study, insertion mutations were detected in two DMN-OMe-induced tumors from this group with previously undefined transforming K-ras alterations. These primary tumors and their NIH3T3 transformants contained K-ras sequences with either a 9 bp or a 12 bp repeat in exon one, both of which included codon 12. No other mutations in the entire coding region of the K-ras gene were observed. Site-directed mutagenesis studies by others have determined that deletions and insertions near codon 12 can activate the ras gene, but this is the first demonstration of insertional activation of K-ras in a chemically induced rat tumor.

Secreted Frizzled-related proteins can regulate metanephric development.

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sfrp-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching were markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restored some tubular differentiation and bud branching. sFRP-2 itself showed no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocked events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it had no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). As shown herein, sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 had no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. On the other hand, sFRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1.

K-ras activation in gastric epithelial tumors in Japanese.

Point mutation in codons 12, 13 and 61 of the K-ras oncogene in gastric epithelial tumors were investigated by polymerase chain reaction from sections of formalin-fixed, paraffin-embedded tissue followed by dot-blot hybridization with mutation-specific oligonucleotide probes. Point mutations were found specifically in four of 20 tumors of intestinal histological subtype; GGT to GAT in three cases and to GTT in one case, all in codon 12 of K-ras. These mutations were also confirmed by direct sequencing. In contrast, none of 11 diffuse-type tumors showed K-ras point mutations. While K-ras point mutations may not be frequent events in gastric tumorigenesis, the similarity of the intestinal-type gastric tumors and colorectal tumors for K-ras point mutations as well as the association of mutations in K-ras with a particular gastric tumor histology implicates K-ras activation in the development of these tumors.

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

Activation of neu by missense point mutation in the transmembrane domain in schwannomas induced in C3H/HeNCr mice by transplacental exposure to N-nitrosoethylurea.

Transplacentally initiated schwannomas in mice and rats arise preferentially in the Gasserian ganglion of the trigeminal nerve and spinal root ganglia, while those of the Syrian golden hamster most commonly occur subcutaneously. Rat and hamster schwannomas almost invariably contain a mutationally activated neu oncogene. In rat schwannomas, the mutant allele predominates, while the relative abundance of mutant alleles is very low in hamster nerve tumors. We investigated whether neu is mutated in mouse schwannomas and whether the pattern and allelic ratio of the mutation resemble those for the hamster or the rat. Pregnant C3H/HeNCr mice received 0.4 micromol N-nitrosoethylurea/g body weight on day 19 of gestation. Ten trigeminal and one peripheral nerve schwannomas developed in 11 of the 201 offspring. Missense T --> A transversion mutations were detected in the neu transmembrane domain in eight of ten schwannomas analyzed, as determined by MnlI digestion of polymerase chain reaction products. The mutant allele was predominantly detected in two tumors and was abundant in six others. Transfection of eight out of ten mouse tumor DNAs into hamster cells yielded transformed foci; seven out of eight contained mutant mouse neu. Mouse schwannomas closely resembled those of rats both in the preferred anatomical site and in the mutant/wild-type neu allele ratios.

[Oncogene neu in spontaneous and induced rat schwannomas: a study using polymerase chain reaction]. / Onkogen neu v spontannykh i indutsirovannykh shvannomakh krys: issledovanie s pomoshch'iu polimeraznoi tsepnoi reaktsii.

Ethylnitrosourea (ENU) given transplacentally to rats induces schwannomas of the cranial, spinal and peripheral nerves with a high frequency of mutation in the neu proto-oncogene. To establish the requirement for such mutation in tumorigenesis of Schwann cells, spontaneous schwannomas from BD-VI strain rats were evaluated for transforming mutations in the transmembrane domain of the encoded protein for the neu proto-oncogene. Whereas all five schwannomas induced by ENU showed T/A transversions in codon 2012 of neu oncogene upon analysis by selective oligonucleotide hybridization and dideoxy sequencing of polymerize chain reaction amplified products from paraffin sections, only one of nine spontaneous schwannomas from untreated rats exhibited the same mutation. Examination of tumours for mutation in codon 12 of Ki-ras proto-oncogene revealed normal alleles. Our conclusions based on these data are that the high frequency of mutations in neu in ENU-induced tumours appears to be attributable to the carcinogen or to the period of development at which exposure occurred, and that transforming mutations of the transmembrane domain of neu, are not required for tumorigenesis of the Schwann cell.

miR-23b targets proline oxidase, a novel tumor suppressor protein in renal cancer.

Proline oxidase (POX) is a novel mitochondrial tumor suppressor that can suppress proliferation and induce apoptosis through the generation of reactive oxygen species (ROS) and decreasing hypoxia-inducible factor (HIF) signaling. Recent studies have shown the absence of expression of POX in human cancer tissues, including renal cancer. However, the mechanism for the loss of POX remains obscure. No genetic or epigenetic variation of POX gene was found. In this study, we identified the upregulated miR-23b in renal cancer as an important regulator of POX. Ectopic overexpression of miR-23b in normal renal cells resulted in striking downregulation of POX, whereas POX expression increased markedly when endogenous miR-23b was knocked down by its antagomirs in renal cancer cells. Consistent with the POX-mediated tumor suppression pathway, these antagomirs induced ROS, inhibited HIF signaling and increased apoptosis. Furthermore, we confirmed the regulation of miR-23b on POX and its function in the DLD1 Tet-off POX cell system. Using a luciferase reporter system, we verified the direct binding of miR-23b to the POX mRNA 3'-untranslated region. In addition, pairs of human renal carcinoma and normal tissues showed a negative correlation between miR-23b and POX protein expression, providing its clinical corroboration. Taken together, our results suggested that miR-23b, by targeting POX, could function as an oncogene; decreasing miR-23b expression may prove to be an effective way of inhibiting kidney tumor growth.

Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53.

Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) induces apoptotic cell death in vitro and in vivo, indicating that it is a promising new therapeutic for cancer. However, the mechanism of its tumor-killing activity remains unclear. Ubiquitin plays an important role in the regulation of tumorigenesis and cell apoptosis. Our earlier report has shown that inactivation of the ubiquitin system through blocking E1 (ubiquitin-activating enzyme) activity preferentially induces apoptosis in p53-expressing transformed cells. As E1 has an active cysteine residue that could potentially interact with NO, we hypothesized that JS-K could inactivate E1 activity. E1 activity was evaluated by detecting ubiquitin-E1 conjugates through immunoblotting. JS-K strikingly inhibits the ubiquitin-E1 thioester formation in cells in a dose-dependent manner with an IC(50) of approximately 2 microM, whereas a JS-K analog that cannot release NO did not affect these levels in cells. Moreover, JS-K decreases total ubiquitylated proteins and increases p53 levels, which is mainly regulated by ubiquitin and proteasomal degradation. Furthermore, JS-K preferentially induces cell apoptosis in p53-expressing transformed cells. These findings indicate that JS-K inhibits E1 activity and kills transformed cells harboring wild-type p53.

Cell adhesion molecules in the kidney: from embryo to adult.

Multiple members from every major family of cell adhesion molecules (CAMs) have been implicated in the development, maintenance, or repair of renal tissues and include several isoforms of integrins, cell-bound glycoproteins, cadherins, immunoglobulin cell adhesion molecules, and selectins. In combination, they mediate a variety of cell-basement membrane and cell-cell interactions believed to direct morphogenesis and cell migration and regulate cell growth and apoptosis, in addition to generating a functional barrier for blood filtration and helping manage inflammatory responses in the kidney. The expression of some CAMs is transient during and critical to normal nephrogenesis, varying with specific stages of development, but often ultimately resulting in the constitutive production of other members in mature tissues. While gene-targeting studies have successfully implicated individual CAMs in renal cell functions, e.g. , alpha3beta1 and alpha8beta1 integrins, the loss of others bears no renal phenotype due to redundancy of homologous family members or to the severity of the defect early in embryogenesis. This review summarizes the studies of various CAMs found in normal embryonic or adult kidney, describes their spatiotemporal expression patterns, and discusses their involvement in renal processes.

Induction of tubules in rat metanephrogenic mesenchyme in the absence of an inductive tissue.

Differentiation of metanephrogenic mesenchyme to renal tubular epithelium requires induction by the ureteric bud in vivo or any of several embryonic tissues in vitro. In an effort to eliminate the tissue requirement in embryonic induction, extracellular matrices and soluble factors were analyzed individually or in combination for their ability to stimulate tubulogenesis in uninduced metanephrogenic mesenchyme from 13-gestation-day rat embryos. These evaluations have established that pituitary extract and epidermal growth factor (EGF) in concert with a matrix can promote morphogenesis of mesenchymal rudiments in culture. While type I collagen, laminin, or fibronectin matrices all promoted tubulogenesis in the presence of pituitary extract and EGF, type IV collagen proved the most effective. Under these conditions, tubules were induced in 23/24 mesenchymal rudiments by 9 days in culture. Mesenchyme was not induced prior to explanation since it formed no tubules when cultured in a medium that allowed tubulogenesis in intact embryonic kidneys. Preliminary characterization of the undefined factor in pituitary extract was consistent with a protein of molecular weight greater than 100,000 but less than 300,000. When uninduced metanephrogenic mesenchyme from mouse was used instead of rat tissue, a similar pattern of morphogenesis was not observed, suggesting that the described medium is inappropriate for promoting differentiation in mouse or, less likely, that different mechanisms mediate differentiation in rat and mouse. These studies show that embryonic induction can occur in explanted rat renal mesenchyme in an appropriate environment and does not require the presence of an inductive tissue.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Growth and branching morphogenesis of rat collecting duct anlagen in the absence of metanephrogenic mesenchyme.

The growth and differentiation of the epithelium in many tissues is mediated by interactions with the adjacent mesenchyme, but the mechanisms responsible remain undefined. To identify the factors involved in the growth and branching morphogenesis of ureteric bud, which is the collecting duct anlagen, buds from 13-gestation-day rat embryos were separated from the metanephrogenic mesenchyme and explanted to culture dishes coated with gelled type I collagen in a defined medium. Under these conditions buds attached to the substrate and grew out without indication of cell senescence. When buds were instead suspended in gelled type I collagen, branching morphogenesis was observed despite the absence of mesenchyme although it was not as extensive as in vivo. Since growth occurred much more slowly in culture than expected, culture conditions were varied in attempts to accelerate the process. Despite extensive screening of matrices and growth factors, only epidermal and endothelial cell growth factors stimulated growth to a significant extent. Transforming growth factor-beta, on the other hand, was a potent inhibitor of growth. Homogenates from tumors that caricature metanephrogenic mesenchyme were highly mitogenic for bud cells and, thus, will be a source of material for characterizing regulatory factors involved in renal growth. These studies show that growth and branching morphogenesis of the ureteric bud can occur without direct cell-cell interactions with the metanephrogenic mesenchyme and that matrices and factors secreted by the mesenchyme may mediated these activities in vivo.

H-ras activation and ras p21 expression in bladder tumors induced in F344/NCr rats by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Bladder tumors were induced in male F344/NCr rats by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at 500 p.p.m. in their drinking water for 12 weeks. Twenty-one bladder tumors that developed between 25 and 50 weeks after BBN administration was begun were evaluated for immunoreactivity with polyclonal or monoclonal antibodies raised against ras p21, for amplification of ras genes by Southern blotting, and for activating point mutations in ras genes by selective oligonucleotide hybridization of products from polymerase chain reaction (PCR). Increased expression of ras p21 was detected by avidin-biotin immunohistochemistry in 18/21 (85%) of the neoplastic bladder lesions. By Southern analysis, there was no significant amplification of H-ras, K-ras or N-ras in any of the tumors except one that showed a 5-fold amplification of K-ras. Point mutations in ras genes were detected by selective oligonucleotide hybridization of the products of PCR. Of the 21 bladder tumors, three tumors were shown to have mutations in codon 12 (GGA----GAA), six tumors in codon 61 (two CAA----CTA, four CAA----CGA), and one in both codon 12 (GGA----GAA) and codon 61 (CAA----CGA), all in H-ras. Thus 10 of 21 tumors has ras gene mutations in a portion of the tumor cells. The variable pattern of point mutation in H-ras suggests that these mutations may not all be a direct consequence of interaction of BBN metabolites with H-ras. Enhanced expression of ras p21 was always focal and was not necessarily associated with transforming ras mutations. It is therefore suggested that tumorigenesis in BBN-initiated bladder cells might involve H-ras activation as part of a multistep pathway; however, H-ras involvement is not obligatory for tumor development.