Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Brca2 is coordinately regulated with Brca1 during proliferation and differentiation in mammary epithelial cells

We have analyzed the expression of the breast cancer susceptibility gene, Brca2, in mammary epithelial cells as a function of proliferation and differentiation. Our results demonstrate that Brca2 mRNA expression is tightly regulated during mammary epithelial proliferation and differentiation, and that this regulation occurs coordinately with Brca1. Specifically, Brca2 mRNA expression is up-regulated in rapidly proliferating cells; is down-regulated in response to serum deprivation; is expressed in a cell cycle-dependent manner, peaking at the G1/S boundary; and is up-regulated in differentiating mammary epithelial cells in response to glucocorticoids. In each case, an identical pattern of expression was observed for Brca1. These results indicate that proliferative stimuli modulate the mRNA expression of these two breast cancer susceptibility genes. In addition, the coordinate regulation of Brca1 and Brca2 revealed by these experiments suggests that these genes are induced by, and may function in, overlapping regulatory pathways involved in the control of cell proliferation and differentiation.

Genetic dissection of dome formation in a mammary cell line: Identification of two genes with opposing action

In this work, we extend the study of the genes controlling the formation of domes in the rat mammary cell line LA7 under the influence of DMSO. The role of the rat8 gene has already been demonstrated. We have now studied two additional genes. The first, called 133, is the rat ortholog of the human epithelial membrane protein 3 (EMP3), a member of the peripheral myelin protein 22 (PMP22)/EMP/lens-specific membrane protein 20 (MP20) gene family that encodes for tetratransmembrane proteins; it is expressed in the LA7 line in the absence of DMSO but not in its presence. The second gene is the β subunit of the amiloride-sensitive Na+ channel. Studies with antisense oligonucleotides show that the formation of domes is under the control of all three genes: the expression of rat8 is required for both their formation and their persistence; the expression of the Na+ channel β subunit is required for their formation; and the expression of gene 133 blocks the expression of the Na+ channel genes, thus preventing formation of the domes. The formation of these structures is also accompanied by the expression of α6β1 integrin, followed by that of E-cadherin and cytokeratin 8. It appears, therefore, that dome formation requires the activity of the Na+ channel and the rat8-encoded protein and is under the negative control of gene 133. DMSO induces dome formation by blocking this control.

Pax6 is essential for establishing ventral-dorsal cell boundaries in pituitary gland development

Pax6, a highly conserved member of the paired homeodomain transcription factor family that plays essential roles in ocular, neural, and pancreatic development and effects asymmetric transient dorsal expression during pituitary development, with its expression extinguished before the ventral → dorsal appearance of specific cell types. Analysis of pituitary development in the Small eye and Pax6 −/− mouse mutants reveals that the dorsoventral axis of the pituitary gland becomes ventralized, with dorsal extension of the transcriptional determinants of ventral cell types, particularly PFrk. This ventralization is followed by a marked decrease in terminally differentiated dorsal somatotrope and lactotrope cell types and a marked increase in the expression of markers of the ventral thyrotrope cells and SF-1-expressing cells of gonadotrope lineage. We suggest that the transient dorsal expression of Pax6 is essential for establishing a sharp boundary between dorsal and ventral cell types, based on the inhibition of Shh ventral signals.

α1 and α2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits α6 and β1, but not against α1 and α2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against β1, but not against α6 or α2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against α1 integrins impaired only cell adhesion to type IV collagen. Antibodies against α1, α2, α6, and β1, but not α5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins α1 and α2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against α1 and α2, but not α6 and β1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against α1 and α2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-α6 antibodies. Our data indicate that α1 and α2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas α6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

WRS-85D: A Tryptophanyl-tRNA Synthetase Expressed to High Levels in the Developing Drosophila Salivary Gland

In a screen for genes expressed in the Drosophila embryonic salivary gland, we identified a tryptophanyl-tRNA synthetase gene that maps to cytological position 85D (WRS-85D). WRS-85D expression is dependent on the homeotic gene Sex combs reduced (Scr). In the absence of Scr function, WRS-85D expression is lost in the salivary gland primordia; conversely, ectopic expression of Scr results in expression of WRS-85D in new locations. Despite the fact that WRS-85D is a housekeeping gene essential for protein synthesis, we detected both WRS-85D mRNA and protein at elevated levels in the developing salivary gland. WRS-85D is required for embryonic survival; embryos lacking the maternal contribution were unrecoverable, whereas larvae lacking the zygotic component died during the third instar larval stage. We showed that recombinant WRS-85D protein specifically charges tRNATrp, and WRS-85D is likely to be the only tryptophanyl-tRNA synthetase gene in Drosophila. We characterized the expression patterns of all 20 aminoacyl-tRNA synthetases and found that of the four aminoacyl-tRNA synthetase genes expressed at elevated levels in the salivary gland primordia, WRS-85D is expressed at the highest level throughout embryogenesis. We also discuss the potential noncanonical activities of tryptophanyl-tRNA synthetase in immune response and regulation of cell growth.

Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Proteomic dissection of dome formation in a mammary cell line: Role of tropomyosin-5b and maspin

In this work we extended the study of genes controlling the formation of specific differentiation structures called “domes” formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the β-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC β-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC β-subunit and the rat8 proteins, and is under the negative control of maspin.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na+], [Cl−], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1–0.5 μl of fluid accumulated in spherical droplets at gland orifices in ≈3–5 min. In gland fluid from normal human airways, [Na+] was 94 ± 8 mM, [Cl−] was 92 ± 12 mM, and pH was 6.97 ± 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 ± 0.3-fold greater than that of saline. Neither [Na+] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by ≈2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na+], [Cl−], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.