Transformation of mouse peritoneal macrophages and bone marrow cells by simian virus 40

A fraction of cultured mouse peritoneal macrophages and bone marrow cells acquired the ability to divide after infection by simian virus 40 (SV40). Two types of transformed lines were obtained. Most transformants isolated 400-60 days after infection did not display macrophage specific properties such as ingestion of opsonized red blood cells, possession of Fc receptors and complement receptors, and acid phosphatase activity throughout the whole culture phase. Cells of the transformed lines isolated by trypsin selection 2--6 months after infection displayed these properties when the cell density became high and cell growth was arrested. In the cells of the latter type of transformed lines, SV40 T-antigen was intensely demonstrated by immunofluorescence in the growing phase, but weakly or not at all in the stationary phase. It is suggested that the reversion to the phenotype of the progenitor (expression of macrophage specific functions) depends on the physiological state of the culture; however, it is uncertain whether the development of the macrophage functions is directly related to the SV40 T-antigen.     

Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Production of interferon by an SV40-transformed macrophage line, BB-W-531-2

A simian virus 40-transformed mouse macrophage line, BB-W-531-2, was examined for its ability to produce interferon. BB-W-531-2 cells showed a phenotypic change between the macrophage and the nonmacrophage states. A viral inhibitor (interferon) was produced by the cells during the phenotypic change from the nonmacrophage to the macrophage state. Cells having macrophage properties were well capable of producing interferon when they were stimulated with ultraviolet-inactivated vaccinia virus, lipopolysaccharide, a streptococcal preparation (OK-432) or polyinosinate . polycytidylate. In contrast, cells that had lost their macrophage properties did not produce interferon even when they were given the same treatments as the cells having macrophage properties. The results suggest that the ability of BB-W-531-2 cells to produce interferon is associated with the expression of several macrophage properties.     

Cloning of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as an anti-apoptotic and growth promoting gene of Burkitt lymphoma cells

Burkitt Lymphoma (BL) cells are highly sensitive to suboptimal growth conditions and undergo apoptosis when seeded at low cell density or reduced serum concentration. Irradiated fibroblasts or a mix of pruvate, alpha-thioglycerol, and bathocuproine disulfonate can protect BL cells from apoptosis induced by lowering cell density or serum concentration by promoting cystine uptake in the cells. The availability of cystine is the limiting factor for glutathione biosynthesis in BL cells and thus for the ability of the cells to cope with oxidative stress. We have set up an expression cloning strategy to clone genes that protect BL cells from apoptosis induced by low cell density and/or serum. Using this approach we have cloned among others the cDNA for Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx).     

Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47(-/-) red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/α2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor-inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47(-/-) red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

CD40 ligand and CTLA-4 are reciprocally regulated in the Th1 cell proliferative response sustained by CD8(+) dendritic cells

Subsets of murine dendritic cells (DCs) from the spleen differ in their ability to induce proliferative responses in both primary and secondary CD4(+) T cells. Recent evidence indicates that lymphoid-related CD8(+) DCs fail to provide appropriate signals to freshly isolated secondary CD4(+) T cells to sustain their proliferation in vitro. In the present study, we examined peptide-pulsed CD8(-) and CD8(+) DCs for ability to stimulate Th1 and Th2 cell clones with the same Ag specificity. Defective ability to induce proliferation was selectively shown by CD8(+) DCs presenting Ag to the Th1 clone. The deficiency in CD8(+) DCs was overcome by CD40 triggering before peptide pulsing. When exposed to CD8(+) DCs in the absence of CD40 activation, the Th1 clone expressed low levels of CD40 ligand and high levels of surface CTLA-4. Neutralization of CTLA-4 during the DC/T cell coculture resulted in increased CD40 ligand expression and proliferation of T cells. Remarkably, the activation of CD40 on DCs under conditions that would increase Th1 cell proliferation, also resulted in down-regulation of surface CTLA-4. These results confirm differential effects of CD8(+) and CD8(-) DCs in the stimulation of Ag-primed Th cells. In addition, they suggest that reciprocal regulation of CD40 ligand and CTLA-4 expression occurs in Th1 cells exposed to CD8(+) DCs.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines.

Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages.     

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. I. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA640 mutant of simian virus 40

It was shown previously that mouse bone marrow cells transformed by simian virus 40 (SV40) show a reversible cell density-dependent phenotypic transition between the nonmacrophage (rapidly growing) and the macrophage (stationary) states; cells in low-density cultures are in the growing phase, express SV40 T antigen strongly as revealed by immunofluorescence, and lose typical macrophage properties such as immune phagocytosis; whereas cells in high-density cultures are in the stationary (nongrowing) phase, express SV40 T antigen weakly, and recover their macrophage properties (Takayama, 1980). In the hope of clarifying the relationship between T antigen, cell growth, and macrophage-specific cellular function, we examined the behavior at 33 and 39 degrees C of mouse bone marrow cells transformed by an SV40 gene A mutant (tsA640) whose mutation renders the molecular weight of 90K (large) T antigen temperature sensitive. The results presented in this paper suggest that functional large T antigen is required for cells in the stationary phase to initiate multiplication when transferred at lower density and is not necessary for a majority of them to maintain the nongrowing state (viability) at both high and lower cell densities, whereas it is required for cells in the growing phase to keep multiplying without losing their viability. The results also suggest that the functional large T antigen does not play a direct role in maintaining the cells as either phagocytic or nonphagocytic. It is also suggested that the physiological or tsA mutation-mediated arrest of growth may or may not be accompanied by induction and/or maintenance of cellular phagocytic activity depending on the culture state.     

Regulation and specificity of MNDA expression in monocytes,macrophages, and leukemia/B lymophoma cell lines

The expression of the human myeloid cell nuclear differentiation antigen (MNDA) was observed specifically in cells of the granulocyte-macrophage lineage in our earlier reports. The specificity of MNDA expression for cells in the granulocyte-macrophage lineage was reexamined in cell line established from patients with philadelphia chromosome-positive chroni myeloid leukemia. Cell lines that expressed MNDA exhibited myeloid cell features and granulocyte or monocyte defferentiation could be induced in vitro, while cell lines exhibiting properties of very early stage cells of multipotential cells ded not express MNDA. Cells orginating from cases of burkitt's lymphoma were negative. By contrast, three Iymphoblastoid cell lines (immortalized in vitro with Epstein-Barr virus) were weakly positive and MNDA was up-regulated by interferon-α (IFN-α) treatment. As we reported previously, MNDA mRNA level in adherent monocytes is elevated by IFN-α; in this study, we further assessed MNDA expression in in vitro monocyte-derived macrophages. Three addditional agents (endotoxin, phytohemagglutinin, and photbol ester) and other conditions that affect function, cutokine production, defferentiation, and/of growth of monocytes were examined for their ability to alter MNDA expression. The results varied with the agent, cell type, and stage of differentiation. Changes in MNDA expression occurred slowly (hours to days), suggesting that MNDA could mediate changes realized over a long period. The results also reveal a discordance in certain MNDA Positiva cells between steady-state levels of changes in levels of protein and mRNA indicating that the regulation of MNDA expression occurs at more than one point. Changes in MNDA expression are consistent with a role in opposing macrophage defferentiation and activation of monocytes/macrophages.     

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.     

Tick saliva regulates migration, phagocytosis, and gene expression in the macrophage-like cell line, IC-21

We studied the effects of tick saliva on cell migration, cell signaling, phagocytosis, and gene expression in the murine macrophage cell line, IC-21. Saliva increased both basal- and platelet-derived growth factor (PDGF)-stimulated migration in IC-21 cells. However, saliva did not affect PDGF-stimulated extracellular signal-regulated kinase (ERK) activity. Zymosan-mediated interleukin-1 receptor associated kinase (IRAK) activity increased when cells were pretreated with saliva. Saliva suppressed phagocytosis of zymosan particles by IC-21 cells. An RT² Profiler™ PCR Array revealed that saliva regulates gene expression in a manner consistent with an immune response skewed toward a Th2 reaction, which is characterized by production of anti-inflammatory cytokines IL-4 and IL-10. Our results using IC-21 cells suggest that Dermacentor variabilis has evolved a mechanism for regulating macrophage function, which may contribute to the tick’s ability to modulate immune function.     

Chromatin Function Modifying Elements in an Industrial Antibody Production Platform - Comparison of UCOE,MAR, STAR and cHS4 Elements

The isolation of stably transfected cell lines suitable for the manufacture of biotherapeutic protein products can be an arduous process relying on the identification of a high expressing clone; this frequently involves transgene amplification and maintenance of the clones’ expression over at least 60 generations. Maintenance of expression, or cell line stability, is highly dependent upon the nature of the genomic environment at the site of transgene integration, where epigenetic mechanisms lead to variable expression and silencing in the vast majority of cases. We have assessed four chromatin function modifying elements (A2UCOE, MAR X_S29, STAR40 and cHS4) for their ability to negate chromatin insertion site position effects and their ability to express and maintain monoclonal antibody expression. Each element was analysed by insertion into different positions within a vector, either flanking or between heavy chain (HC) and light chain (LC) antibody expression cassettes. Our results clearly show that the A2UCOE is the most beneficial element in this system, with stable cell pools and clones increasing antibody yields 6.5-fold and 6.75-fold respectively. Stability analysis demonstrated that the reduction in antibody expression, seen with cells transfected with the control vector over 120 generations, was mitigated in the clones containing A2UCOE-augmented transgenes. Analysis also showed that the A2UCOE reduced the amount of transgene promoter DNA methylation, which contributed to the maintenance of starting levels of expression.     

Role of cell density/cell-cell contact,and growth state in expression of differentiated properties by the LLC-PK1 Cell

Populations of the renal epithelial cell line, LLC-PK,₁, acquire many properties characteristic of the proximal tubular cell at confluence. At confluence cells both enter a nonproliferative state and develop extensive cell-cell contacts. To determine if one or both factors is responsible for acquisition of the differentiated phenotype, growth arrest was initiated in populations of varying densities by two procedures (serum deprivation and thymidine block) and expression of several differentiated properties (Na-hexose symport activity, gamma-glutamyl transpeptidase activity, alkaline phosphatase activity, and villin protein) was examined. Induction of growth arrest resulted in expression of all differentiated properties even in subconfluent populations. The level of expression in a population was proportional to cell density at the initiation of growth arrest; higher density was associated with increased expression. Evidence indicated the existence of some minimal density below which cells could not express detectable levels of differentiated properties in response to induction of growth arrest. The procedure used to initiate growth arrest did not affect this behavior, indicating that initiation of cell growth arrest rather than hormone deprivation was the inducing factor. These results indicate that both cell growth state and cell density independently modulate expression of differentiated properties by the LLC-PK₁ cell. These results are incorporated into a model in which cells in the absence of “appropriate” cell-cell contact arrest at a differentiation-incompetent cell cycle point. In the presence of appropriate cell-cell contact (as yet undefined) cells arrest at a distinct differentiation-competent cell cycle point and initiate expression of the differentiated phenotype. © 1994 wiley-Liss, Inc.     

Macrophage cell lines derived from major histocompatibility complex II-negative mice

Summary Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II-negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-γ, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphylococcal enterotoxins A or B. C2Dt cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

双氢青蒿素调控巨噬细胞增殖和迁移的作用研究

目的:探讨双氢青蒿素在体外对小鼠单核巨噬细胞RAW264.7的增殖、克隆形成、周期、凋亡和迁移的影响。方法:采用梯度浓度(2.5μg/m L, 5μg/m L, 10μg/m L, 20μg/m L)的双氢青蒿素处理RAW264.7细胞,利用CCK8实验检测双氢青蒿素对巨噬细胞增殖能力的影响,利用克隆形成实验检测双氢青蒿素对RAW264.7细胞克隆形成能力的影响,利用流式细胞术检测双氢青蒿素对RAW264.7细胞周期和凋亡的影响,利用划痕修复实验检测RAW264.7细胞迁移能力。结果:CCK8实验结果显示,双氢青蒿素可以显著抑制RAW264.7巨噬细胞的增殖能力,且抑制效果与双氢青蒿素的浓度呈正相关性。克隆形成实验结果显示,双氢青蒿素可以抑制细胞的克隆形成能力。双氢青蒿素处理使RAW264.7细胞G0/G1期比例显著升高,S期与G2/M期细胞比例显著降低。双氢青蒿素对巨噬细胞凋亡具有诱导作用,且凋亡诱导作用呈现浓度依赖的特性。划痕修复实验结果显示,双氢青蒿素可以显著抑制RAW264.7巨噬细胞的迁移能力。结论:双氢青蒿素可以导致巨噬细胞的细胞周期G0/G1阻滞,并且诱导细胞凋亡,对巨噬细胞增殖和迁移具有抑制作用。     

PHLDA1 (pleckstrin homology-like domain,family A,member 1) knockdown promotes migration and invasion of MCF10A breast epithelial cells

PHLDA1 (pleckstrin homology-like domain, family A, member 1) is a multifunctional protein that plays distinct roles in several biological processes including cell death and therefore its altered expression has been identified in different types of cancer. Progressively loss of PHLDA1 was found in primary and metastatic melanoma while its overexpression was reported in intestinal and pancreatic tumors. Previous work from our group showed that negative expression of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer disease. However, the function of PHLDA1 in mammary epithelial cells and the tumorigenic process of the breast is unclear. To dissect PHLDA1 role in human breast epithelial cells, we generated a clone of MCF10A cells with stable knockdown of PHLDA1 and performed functional studies. To achieve reduced PHLDA1 expression we used shRNA plasmid transfection and then changes in cell morphology and biological behavior were assessed. We found that PHLDA1 downregulation induced marked morphological alterations in MCF10A cells, such as changes in cell-to-cell adhesion pattern and cytoskeleton reorganization. Regarding cell behavior, MCF10A cells with reduced expression of PHLDA1 showed higher proliferative rate and migration ability in comparison with control cells. We also found that MCF10A cells with PHLDA1 knockdown acquired invasive properties, as evaluated by transwell Matrigel invasion assay and showed enhanced colony-forming ability and irregular growth in low attachment condition. Altogether, our results indicate that PHLDA1 downregulation in MCF10A cells leads to morphological changes and a more aggressive behavior.