Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 109 records · Page 6Linked to original sources

PubMed1993

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells.

Alternative Splicing↗

PubMed1993

Benign prostatic hyperplasia in a transgenic mouse: a new hormonally sensitive investigatory model.

Recent advances in molecular biology have enabled incorporation of proto-oncogenes into the mouse germline. In this study we use a transgenic mouse line that overexpresses the fibroblastic growth factor (FGF) family member, int-2, under the control of mouse mammary tumor virus (MMTV) regulatory elements. One of the tissues targeted by MMTV is the mouse prostate. Expression of the MMTV-int-2 transgene in male transgenic mouse carriers results in a dramatic enlargement of the prostate gland which on histologic examination closely resembles the epithelial/glandular BPH observed in human and canine models. Pre- and postpubertal transgenic (NR) and wild-type (WT) FVB/N male mice were evaluated for the effects of hormonal manipulation by orchiectomy and orchiectomy followed by androgen replacement. Orchiectomy results in a significant decrease in size of the prostate in both NR and WT mice (p < 0.05), regardless of sexual maturity. Exogenous hormonal replacement with testosterone or dihydrotestosterone following orchiectomy results in significant regrowth of the prostate in both NR and WT mice. Flutamide, a potent nonsteroidal anti-androgen, resulted in a 55% reduction in size of the NR prostate (p < 0.002) and a similar 44% reduction in size of the WT prostate. Similarly, treatment of both NR and WT mice with leuprolide, a GnRH agonist, resulted in a significant decrease in prostate size (p < 0.05). Treatment of both NR and WT mice with finasteride (MK-906), a 5-alpha reductase inhibitor, failed to produce any significant regression in prostatic tissue. Based upon these data, we conclude that this transgenic mouse model, expressing int-2, produces an epithelial BPH histologically similar to other animal models. This transgenic model is hormonally sensitive and appears to represent a unique model for the investigation of BPH and growth factor induced epithelial cell hyperplasia.

5-alpha Reductase Inhibitors↗

PubMed1993

Multiple tumor types appear in a transgenic mouse with the ras oncogene.

A transgenic mouse strain with the zeta-globin promoter and the vHa-ras oncogene develops an array of mesenchymal and epithelial neoplasms described here. The predominate mesenchymal tumors were dermal spindle cell tumors, which resembled malignant fibrous histiocytomas found in humans. They were associated with hepatosplenomegaly and developed beneath squamous papillomas. The hepatosplenomegaly was associated with infiltrates of cells that tended toward myelocytic or monocytic differentiation. Other epithelial tumors included keratoacanthomas and squamous cell carcinomas. Squamous cysts, some with squamous cell carcinomas, of the salivary glands and mammary carcinomas were also found. Odontogenic tumors, which sometimes differentiated into ameloblastomas, were one of the more unusual tumor types observed. Other, less frequent tumors were also noted. The tumors described here are a potentially valuable experimental resource that may lead to an understanding of malignant fibrous histiocytoma-like lesions, odontogenic tumors, and tumor progression.

Animals↗

PubMed1992

Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro.

Among its many activities, leukemia inhibitory factor (LIF) can maintain embryonic stem cell monolayers in a pluripotent undifferentiated state. Presuming that this might reflect its physiologic role during embryogenesis, we have examined LIF expression in the embryonic environment by RNase protection assays and have determined its in vitro effect on differentiating embryonic stem cell embryoid bodies. Of all adult tissues analyzed, LIF transcripts appear only in the uterus, where their level fluctuates with the estrous cycle, peaking after ovulation. LIF expression continues in the uteri of pregnant and pseudopregnant females, with a relative peak when blastocysts are normally present. As for its effects on in vitro differentiation, we have found that LIF blocks embryoid body differentiation only partially, yet in a precise manner. Using molecular markers to follow the differentiation of defined cell types, we demonstrate that LIF selectively inhibits the formation of primitive ectoderm, while permitting the differentiation of primitive endoderm. These results suggest a specific role for LIF in preimplantation mouse development.

Animals↗

PubMed1992

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.

Amino Acid Sequence↗

PubMed1992

An eosinophil-dependent mechanism for the antitumor effect of interleukin-4.

Murine interleukin-4 (IL-4) exhibits potent antitumor activity when present at the site of tumor cell challenge. Associated with tumor cell death is the appearance of an inflammatory infiltrate comprised predominantly of eosinophils and macrophages, but with few lymphocytes. Antibodies that specifically block the accumulation of granulocytes at the site of inflammation were injected in vivo to define the cell type responsible for the antitumor action of IL-4. These studies implicate eosinophils in IL-4-mediated tumor cytotoxicity. The lymphoid-independent nature of IL-4 action is supported by the analysis of mutant mouse strains with defined lymphocyte immunodeficiencies. The observed regression of established tumor masses by localized IL-4 action provides a rationale for exploring IL-4-mediated tumor killing as a potential therapy for human malignant disorders.

Animals↗

PubMed1992

A receptor binding domain of mouse interleukin-4 defined by a solid-phase binding assay and in vitro mutagenesis.

Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.

Amino Acid Sequence↗

PubMed1992

Int-2, an autocrine and/or ultra-short-range effector in transgenic mammary tissue transplants.

BACKGROUND: Previous studies have shown associations between overexpression of int-2 messenger RNA (mRNA) and murine mammary tumors and between amplification of the int-2 genomic locus and human breast cancers. The Int-2 protein (fibroblast growth factor 3) is a member of the heparin-binding growth factor family of proteins. The export of these growth factors from cells may depend on the presence of amino terminal sequences containing hydrophobic signal peptides. Although Int-2 has a putative signal sequence, it is not known whether or how this protein is secreted from cells. PURPOSE: Assuming that the Int-2 protein is secreted from mammary epithelial cells in a basolateral direction so that it is available to affect adjacent cells, we investigated whether it acts in a paracrine manner, exerting its effect externally on adjacent cells, or in an autocrine manner, exerting its effect internally within the same cell. METHODS: Using in situ hybridization with 35S-labeled RNA antisense probes that specifically detect mRNA coding for the Int-2 protein, we determined the cell-specific localization of int-2 mRNA expression in the mammary gland of transgenic FVB/N mice overexpressing int-2 mRNA. Then we transplanted pieces of mammary epithelial tissue expressing int-2 mRNA into the mammary fat-pad of wild-type, syngeneic animals. The mammary glands of host animals were examined as whole-mounts and as histologic sections 2-6 months after transplantation. In situ hybridization was used to confirm which cells continued to express int-2 mRNA following transplantation. RESULTS: Int-2 mRNA expression in transgenic mice was localized to the mammary epithelial cells. Transplants expressing int-2 mRNA were similar to wild-type transplants in that they had no observable effect on either the growth or the morphology of host mammary epithelium. Abnormal growth occurred only in transplanted tissue expressing int-2 mRNA but not in adjacent host mammary epithelium. CONCLUSION: Given the limitations of our experimental system and the limited information available to date on the secretion of Int-2 protein, these results suggest that, although the Int-2 protein contains a putative signal peptide, it may act primarily as an autocrine or as an ultra-short-range paracrine growth factor in mammary epithelium.

Animals↗

PubMed1992

B-less: a strain of profoundly B cell-deficient mice expressing a human lambda transgene.

We have created several transgenic mouse strains that bear the human lambda light chain gene driven by its own promoter and a mouse immunoglobulin heavy chain enhancer. The transgene is expressed in many tissues, with particularly high levels of expression in the bone marrow, thymus, spleen, and lymph nodes. One of these transgenic lines, B-less, displays a dramatic phenotype characterized by an acute susceptibility to bacterial and viral infections. Analysis of this strain shows it to be profoundly deficient in both immature (pre-B) and mature B cells, as well as in circulating immunoglobulin. The pre-B and B cell defects are cell autonomous, as judged by cell culture and bone marrow graft chimeras. Despite this B cell deficiency, the T cell lineage appears grossly normal as assessed by flow cytometric analysis and by its response to mitogen stimulation. Since an independently derived transgenic strain bearing the same human lambda construct displays a partial B-less phenotype, it is likely that the B lineage deficiency is due to a dominant effect of transgene expression rather than to the insertional perturbation of an endogenous mouse gene. It is interesting that the deficiency phenotype is fully expressed in the FVB/N genetic background, but is suppressed in F1 hybrids formed between the FVB/N and C57BL/6 inbred strains. Evidently, there are one or more dominant genetic suppressors of B-less in the C57BL/6 genome.

Animals↗

PubMed1992

The same genomic region is disrupted in two transgene-induced limb deformity alleles.

Mutations of the mouse limb deformity locus, ld, map to Chromosome (Chr) 2 and result in defects in the morphogenesis and patterning of the limb and kidney. Complementation studies have defined the existence of five recessive ld alleles. Remarkably, two of these, ldTgHd and ldTgBri, are transgene-induced mutations. Recovery of the first transgene insertional allele, ldTgHd, facilitated the molecular cloning of a large (greater than 200 kb) candidate gene at the ld locus. This gene is broadly transcribed and encodes a set of novel protein isoforms, termed formins. Here we present characterization of the ldTgBri mutation that supports the molecular identification of the ld gene. We show that the ldTgBri fails to complement both the ldTgHd and the ldOR alleles and that it has undergone a genomic deletion that disrupts the cloned ld gene and its transcripts. Curiously, the ldTgBri deletion encompasses the same 11-kb interval in which the ldTgHd insertion occurred and in which a chromosomal rearrangement has been identified in a third allele, ldIn2. These findings suggest that this region of the ld gene is a preferential site for illegitimate recombination.

Alleles↗

PubMed1992

A variant limb deformity transcript expressed in the embryonic mouse limb defines a novel formin.

The formins constitute a set of protein isoforms encoded by the alternatively spliced transcripts arising from the limb deformity (ld) locus of the mouse. Mutations in this locus disrupt formation of the anteroposterior axis of the embryonic limb. Although ld transcripts are widely expressed during embryogenesis, we have identified a novel transcript that is expressed in the mesenchyme and apical ectodermal ridge of the developing limb. This pattern of expression coincides with the earliest morphological defects observed in ld mutant limb buds. Moreover, the formin encoded by this transcript bears a highly acidic amino terminus, as distinguished from the basic amino terminus encoded by other ld transcripts suggesting that it may have a distinct biochemical function.

Amino Acid Sequence↗

PubMed1992

An embryonically expressed gene is a target for c-Myc regulation via the c-Myc-binding sequence.

We have used a subtraction/coexpression strategy involving two different tumors derived from c-myc-bearing transgenic mice to identify a gene that is a target for c-Myc regulation. The gene, expressed in certain embryonic and adult tissues and in several (but not all) c-myc-based tumors, bears a functional c-Myc-binding sequence located 3' to its transcription start site. This sequence is required for the binding of a nuclear protein complex which, by antibody analysis, includes c-Myc. This site is also required for expression of a reporter gene in chimeric constructs transfected into c-myc-overexpressing cells and, conversely, requires c-myc cotransfection for its enhanced expression in COS cells. Furthermore, transfection of c-myc blocks the normal down-regulation of this gene, which occurs in embryonic stem cells as they undergo differentiation. This target gene encodes an anonymous cDNA (ECA39) found previously to be amplified in a teratocarcinoma cell line.

Animals↗

PubMed1992

Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice.

The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.

Animals↗

PubMed1992

Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.

Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.

3T3 Cells↗

PubMed1992

In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors.

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

Animals↗

PubMed1992

Brain tumours and lymphomas in transgenic mice that carry HTLV-I LTR/c-myc and Ig/tax genes.

The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.

Animals↗

PubMed1991

Humoral immune functions in IL-4 transgenic mice.

We have analyzed mice expressing IL-4 as a transgene, and found that expression of this lymphokine has profound effects on B cell function. B cells from transgenic mice exhibit phenotypic changes, including an increase in size and elevated expression of class II MHC. IL-4 increases the quantity of IgE produced by transgenic-derived B cells in response to LPS stimulation. In vivo, IL-4 markedly affects the serum Ig isotype repertoire. Serum levels of IgG1 and IgE are elevated, and levels of IgG2a, IgG2b, and IgG3 are depressed in IL-4 transgenic mice. Ag-specific antibody responses to immunization with hapten-carrier conjugates are also affected by IL-4. Transgenic mice show increased anti-hapten IgE and IgG1 and reduced anti-hapten IgG2a, IgG2b, and IgG3, compared with wild-type mice. Ag-specific IgE is substantially induced by T cell-dependent Ag, but not T cell-independent Ag, suggesting that cognate T-B interactions in addition to IL-4 are required for generating IgE responses in vivo. In vivo treatment with the anti-IL-4 mAb 11B11 reverses many of the isotype alterations in the transgenic mice, indicating that these changes arise as a direct consequence of IL-4 secretion.

Animals↗

PubMed1991

In vitro and in vivo analysis of the c-myc RNA polymerase III promoter.

The c-myc promoter has the unusual property of displaying both RNA polymerase II (Pol II) and RNA polymerase III (Pol III) activities. Both Pol II and Pol III utilize the same transcription initiation site. We have now examined the effects of mutations in crucial regions of the c-myc promoter to assess their effects on both transcriptional activities. In doing this we show that both Pol II and Pol III activities require sequences that are located within the stronger of the two principal c-myc promoter regions (P2). Further, we show that the Pol III activity using this initiation site does not require an A box or distal upstream sequences. Like the Pol II activity, it does require an intact TATA sequence and alterations at this site result in the simultaneous loss of both Pol II and Pol III activities. The superimposition of two apparently inseparable promoter activities makes it possible to consider common features, possible common protein elements in each holoenzyme complex, as well as a potential role for each enzyme in the regulated expression of the c-myc gene.

Base Sequence↗