Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells.

Mouse 3T3 cells were transformed with an antisense c-fos gene fused to a mouse mammary tumor virus promoter. In transformants that integrated a large number of antisense c-fos sequences, the usual large increase in c-fos mRNA and protein following stimulation of quiescent cells by platelet-derived growth factor was blocked in the presence of dexamethasone. These cells subsequently also failed to show the stimulation of DNA synthesis normally induced by platelet-derived growth factor. Appropriate expression of c-fos appears to be a prerequisite for reentry of quiescent cells into the cell cycle.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.     

Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.     

Antisense expression of a cell wall-associated protein kinase, WAK4, inhibits cell elongation and alters morphology

The Arabidopsis cell wall-associated receptor-like kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the intracellular and extracellular compartments. WAK members are expressed in specific organs and regulated differentially by various biotic and abiotic factors. To gain further insight into how WAKs function during development, we used a glucocorticoid-inducible system to express ectopically the WAK4 antisense gene. The induced expression of the WAK4 antisense gene resulted in a significant decrease of WAK proteins. Ninety-six hours after the induction of WAK4 antisense expression, WAK proteins became undetectable. Cell elongation was impaired, and lateral root development was blocked. The level of WAK protein could be controlled by the concentration of the applied inducer, dexamethasone, and was correlated with the severity of the cell elongation inhibition phenotype. These results suggest that the WAKs serve a vital role in cell elongation and are required for plant development.     

Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodriguez Fernandez, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zoller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.     

An Arabidopsis cell cycle -dependent kinase-related gene, CDC2b, plays a role in regulating seedling growth in darkness.

The Arabidopsis CDC2b gene has been defined as a plant-specific cell cycle-dependent kinase-related gene, although it lacks the conserved cyclin binding motif, and its exact function is not known. Here, we report that in etiolated seedlings, the expression of the CDC2b gene is correlated with elongation rate of the hypocotyl. Inhibition of CDC2b gene expression by using an inducible antisense construct resulted in short-hypocotyl and open-cotyledon phenotypes when transgenic seedlings were grown in the dark. The severity of these phenotypes in dark-grown seedlings could be correlated with the level of the antisense gene expression. The short hypocotyl of seedlings underexpressing CDC2b was a result of inhibition of cell elongation rather than a reduction in cell number, whereas in cotyledons, inhibition of CDC2b expression resulted in large, open cotyledons with amyloplasts rather than etioplasts. Although the nuclear DNA was less compact in the antisense hypocotyl cells, DNA content and endoreduplication were not affected. Cell division of the shoot apical meristem also was not affected by antisense expression. The short-hypocotyl phenotype of these transgenic plants was partially rescued by the addition of brassinolide. Brassinolide can only induce CDC2b expression in darkness. These results suggest a role for the CDC2b gene in seedling growth via regulation of hypocotyl cell elongation and cotyledon cell development.     

Antisense Expression of a Cell Wall–Associated Protein Kinase, WAK4, Inhibits Cell Elongation and Alters Morphology

The Arabidopsis cell wall–associated receptor-like kinase (WAK) gene family contains five highly related members whose products are suited for exchanging signals between the intracellular and extracellular compartments. WAK members are expressed in specific organs and regulated differentially by various biotic and abiotic factors. To gain further insight into how WAKs function during development, we used a glucocorticoid-inducible system to express ectopically the WAK4 antisense gene. The induced expression of the WAK4 antisense gene resulted in a significant decrease of WAK proteins. Ninety-six hours after the induction of WAK4 antisense expression, WAK proteins became undetectable. Cell elongation was impaired, and lateral root development was blocked. The level of WAK protein could be controlled by the concentration of the applied inducer, dexamethasone, and was correlated with the severity of the cell elongation inhibition phenotype. These results suggest that the WAKs serve a vital role in cell elongation and are required for plant development.     

Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation.

Cell differentiation is associated either with a complete loss of proliferative potential or with a change in growth requirements. Neoplastic transformation may result from the activation of oncogenes that support growth or from inactivation or loss of tumor suppressor genes, which are thought to regulate differentiation. To examine the relationship between tumor suppressor genes and cell differentiation, we chose the gene "deleted in colorectal cancer" (DCC) and studied its role in a pheochromocytoma cell line, PC-12, using antisense RNA as well as antisense oligonucleotides to DCC. When exposed to nerve growth factor for several days, PC-12 cells develop long dendrites. This morphological change follows the transient expression of immediate early genes and is associated with an up-regulation of DCC. Interestingly, if the up-regulation of DCC was counteracted using an antisense RNA technique, the morphological changes were prevented, but the other parameters of the nerve growth factor response were unaffected. Moreover, when DCC expression was inhibited by antisense oligonucleotides to DCC in nerve growth factor-differentiated cells, the neuron-like phenotype was reversed. Our results demonstrate that the gene DCC is involved in a distal segment of neural differentiation and provide the first direct evidence that a tumor suppressor gene plays a role in cell differentiation.     

Rap1 Overexpression Reveals That Activated RasD Induces Separable Defects duringDictyosteliumDevelopment

One of theDictyostelium rasgenes,rasD,is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymondet al.,1986,Nature,323, 340–343). The ability of theDictyostelium rap1gene to suppress this abnormal developmental phenotype was investigated. Therap1gene and G12V activated and G10V negative mutant forms of therap1gene were independently linked to therasDpromoter and each construct used to transform M1, aDictyosteliumcell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). Therap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louiset al.,1997,Mol. Biol. Cell,8, 303–312). During the development of ME cells,ecmA mRNA levels were restored to those seen for Ax3, andtagB mRNA levels were also markedly reduced, although not to Ax3 levels.cotCexpression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression ofcar1mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed byrap1.     

Outward-rectifying K+ channel activities regulate cell elongation and cell division of tobacco BY-2 cells

Potassium ions (K⁺) are required for plant growth and development, including cell division and cell elongation/expansion, which are mediated by the K⁺ transport system. In this study, we investigated the role of K⁺ in cell division using tobacco BY-2 protoplast cultures. Gene expression analysis revealed induction of the Shaker -like outward K⁺ channel gene, NTORK1 , under cell-division conditions, whereas the inward K⁺ channel genes NKT1 and NtKC1 were induced under both cell-elongation and cell-division conditions. Repression of NTORK1 gene expression by expression of its antisense construct repressed cell division but accelerated cell elongation even under conditions promoting cell division. A decrease in the K⁺ content of cells and cellular osmotic pressure in dividing cells suggested that an increase in cell osmotic pressure by K⁺ uptake is not required for cell division. In contrast, K⁺ depletion, which reduced cell-division activity, decreased cytoplasmic pH as monitored using a fluorescent pH indicator, SNARF-1. Application of K⁺ or the cytoplasmic alkalizing reagent (NH₄)₂SO₄ increased cytoplasmic pH and suppressed the reduction in cell-division activity. These results suggest that the K⁺ taken up into cells is used to regulate cytoplasmic pH during cell division.     

Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes.     

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.     

A cell wall protein down-regulated by auxin suppresses cell expansion in Daucus carota (L.)

We investigated the function of the auxin-regulated cell wall gene DC 2.15, a member of a small gene family, present in Daucus carota (L.) and other plants. Cultured cells derived from carrot hypocotyls transformed by the DC 2.15 cDNA in antisense direction were ten-fold longer than wild-type cells, indicating a function of the corresponding protein in suppression of cell expansion. The analysis of carrot plants expressing the DC 2.15 gene in antisense direction showed that the corresponding protein and/or related proteins probably are involved in leaf and vascular bundle development. The antisense plants generally displayed a retarded growth phenotype and delayed greening in comparison to wild-type plants. The asymmetric architecture of the wild-type leaves was degenerated in the DC 2.15 antisense plants and the leaves showed a torsion within and along their major vein. The vascular bundles showed a lowered ratio of the phloem/xylem area in cross sections of the leaf middle vein whereas the bundle sheath and the cambium showed no obvious phenotype. Expression of a promoter-GUS construct was found primarily in vascular bundles of stems, leaves and in the nectar-producing flower discs. The observed pleiotropic antisense phenotype indicates, by loss of function, that one or several related cell wall proteins of this gene family are necessary to realize several complex developmental processes.