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Dominant Negative (dominant + negative)

Distribution by Scientific Domains

Life Sciences	50%
Medical Sciences	43%

Terms modified by Dominant Negative

dominant negative effect

dominant negative form

dominant negative i

dominant negative mutant

dominant negative ras

Selected Abstracts

Gene Transfer of TRPC6DN (Dominant Negative) Restores Erectile Function in Diabetic Rats

THE JOURNAL OF SEXUAL MEDICINE, Issue 3 2010
Jae Hun Jung MD
ABSTRACT Introduction., Transient receptor potential (TRP) channels play an important role in modulating intracellular Ca2+ ([Ca2+]i) levels. Aim., We examined the hypothesis that overexpression of TRPC6DN (dominant negative) may contribute to decreased [Ca2+]i levels in corporal smooth muscle (CSM). We also investigated whether gene transfer of TRPC6DN could restore erectile function in diabetic rats. Methods., For the in vitro study, the KCa, KATP, and TRPC6DN channel genes were transferred using cDNA, into cultured human CSM cells and human embryonic kidney cells. For the in vivo study, young adult rats were divided into three groups: normal controls; diabetic controls transfected with vector only; and a diabetic group transfected with pcDNA of the TRPC6DN gene. Main Outcome Measures., After gene transfer, the effects of reducing [Ca2+]i levels were assessed by Fura-2-based imaging analysis. The intracavernosal pressure (ICP) response to cavernosal nerve stimulation was assessed after intracorporal injection of TRPC6DN pcDNA. The transgene expression of the TRPC6DN was examined by reverse transcription polymerase chain reaction (RT-PCR) in rats transfected with TRPC6DN pcDNA. Results., Gene transfer of ion channels effectively reduced [Ca2+]i. Among these channels, transfer of the TRPC6DN gene resulted in the greatest reduction of [Ca2+]i in human CSM. The mean (±standard error of the mean) ratio of ICP to mean arterial pressure (BP) in the gene-transfer rats was 79.4 ± 2.4% (N = 8). This was significantly higher than that in control rats (55.6 ± 3.7% [N = 8]), and similar to that in the young control rats (83 ± 2.2% [N = 12]). The RT-PCR showed expression of TRPC6DN genes in the transfected rats. Conclusion., Gene transfer of TRPC6DN not only reduced [Ca2+]i in human CSM but also restored erectile function in diabetic rats. These results suggest that pcDNA transfer of TRPC6DN may represent a promising new form of therapy for the treatment of male erectile dysfunction in the future. Jung JH, Kim BJ, Chae MR, Kam SC, Jeon J-H, So I, Chung KH, and Lee SW. Gene transfer of TRPC6DN (dominant negative) restores erectile function in diabetic rats. J Sex Med 2010;7:1126,1138. [source]

Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma,

THE LARYNGOSCOPE, Issue 12 2004
Joseph C. Sniezek MD
Abstract Objectives/Hypothesis: p63, a member of the p53 family of genes, is vital for normal epithelial development and may play a critical role in epithelial tumor formation. Although p63 has been identified in various head and neck malignancies, a detailed analysis of which of the six isoforms of the p63 gene is present in normal mucosa and head and neck malignancies has not yet been performed. The study analyzed p63 isoform expression on the RNA and protein level in normal, diseased, and malignant mucosa of the head and neck to examine the differential expression of p63 isoforms in head and neck tumors versus adjacent nonmalignant tissue and to identify the predominant p63 isoform expressed in head and neck squamous cell carcinoma (HNSCC). Study Design: Three experiments were performed. In experiment 1, p63 expression was analyzed by immunohistochemical analysis in 36 HNSCC specimens and matched normal tissue control specimens harvested from the same patient. Western blot analysis was also performed on matched specimens to confirm the identity of the p63 isoforms that were found. In experiment 2, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on matched normal and tumor specimens to analyze and quantitatively compare p63 isoform expression at the RNA level. In experiment 3, p63 expression was evaluated by immunohistochemical analysis in oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. Methods: Immunohistochemical analysis, RT-PCR, and Western blot analysis of p63 were performed on HNSCC specimens and matched normal tissue control specimens. p63 expression in oral lichen planus specimens was also examined by immunohistochemical analysis. Results: In experiment 1, analysis of 36 HNSCC specimens from various head and neck subsites showed p63 expression in all tumors and matched normal tissue specimens (36 of 36). Western blot analyses indicated that dominant negative (,N) isoform p63, (,Np63,) is the major isoform expressed at the protein level in tumors and adjacent normal tissue. In experiment 2, RT-PCR analyses of 10 matched specimens confirmed that, although all three ,Np63 isoforms (,Np63,, ,Np63,, and ,Np63,) are expressed in normal and malignant mucosa of the head and neck, ,Np63, is the predominant transcript expressed. In experiment 3, immunohistochemical analysis of p63 in the pro-apoptotic condition of lichen planus indicated that p63 is underexpressed as compared with normal mucosal specimens. Conclusion: Although all three ,Np63 isoforms are present in HNSCC, ,Np63, protein is the predominant isoform expressed in these malignancies. ,Np63, is also overexpressed in tumors compared with matched normal tissue specimens and is underexpressed in the pro-apoptotic condition of lichen planus. These findings suggest that ,Np63, plays an anti-differentiation and anti-apoptotic role in the mucosal epithelium of the head and neck, possibly playing a pivotal role in the formation of HNSCC. Currently, ,Np63, is an attractive target for mechanistic study aimed at therapeutic intervention. [source]

R-Ras promotes tumor growth of cervical epithelial cells

CANCER, Issue 3 2003
Héctor Rincón-Arano B.S.
Abstract BACKGROUND R-Ras is 55% identical to H-Ras. However, these two oncogenes seem to have different tumor-transforming potential. R-Ras induced cell transformation in fibroblasts but not in other cell types. R-Ras also reportedly induces a more invasive phenotype in breast epithelial cells through integrin activation. The authors studied the mechanisms whereby R-Ras induces a malignant phenotype. METHODS Dominant negative (R-Ras43N) and constitutively active (R-Ras87L) mutants of R-Ras were stably transfected into human cervical epithelium C33A cells. Transfected cells were analyzed for adhesion, cell spreading, migration, and growth in culture and in nude mice. The activity of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K) also was determined by Western blot analysis and by in vitro kinase assays. RESULTS R-Ras87L-transfected cells, but not R-Ras43 N-transfected cells, had a higher growth rate in nude mice and in culture compared with control cells. None of the transfected C33A cells showed an increase in cell adhesion to fibronectin or collagen I, nor did they show an increment of ,1 integrin affinity. However, cells that expressed R-Ras87L, but not cells that expressed R-Ras 43N, presented a marked increase in cell spreading and migration through collagen-coated membranes. Increases in cell proliferation, spreading, and migration induced by R-Ras87L were inhibited by the PI 3-K inhibitor LY294002. In addition, PI 3-K activity, but not ERK activity, was increased only in cells that expressed R-Ras87L. CONCLUSIONS These data suggest that the oncogene R-Ras promotes tumor growth of cervical epithelial cells and increases their migration potential over collagen through a pathway that involves PI 3-K. Cancer 2003;97:575,85. © 2003 American Cancer Society. DOI 10.1002/cncr.11093 [source]

The unconventional myosin-VIIa associates with lysosomes

CYTOSKELETON, Issue 1 2005
Lily E. Soni
Abstract Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor. Cell Motil. Cytoskeleton 62:13,26, 2005. © 2005 Wiley-Liss, Inc. [source]

Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanism

DEVELOPMENTAL DYNAMICS, Issue 4 2004
Gary R. Hime
Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source]

INSULIN-LIKE GROWTH FACTOR-I RECEPTOR AS A CANDIDATE FOR A NOVEL MOLECULAR TARGET IN GASTROINTESTINAL CANCERS

DIGESTIVE ENDOSCOPY, Issue 4 2006
Yasushi Adachi
Abnormal activation of growth factor receptors and their signal pathways are required for neoplastic transformation and tumor progression. The concept of targeting specific tumorigenic receptors has been validated by successful clinical application of multiple new drugs, such as those acting against HER2/neu, epidermal growth factor receptor 1, and c-Kit. In this review, we focus on the next promising therapeutic molecular target of insulin-like growth factor (IGF)-I receptor (IGF-Ir). The IGF/IGF-Ir system is an important modifier of cancer cell proliferation, survival, growth, and treatment sensitivity in a number of neoplastic diseases, including human gastrointestinal carcinomas. Preclinical studies demonstrated that downregulation of IGF-Ir signals reversed the neoplastic phenotype and sensitized cells to antitumor treatments. We summarize a variety of ways to disrupt IGF-Ir function. Then, we introduce our strategy of adenoviruses expressing dominant negative of IGF-Ir (IGF-Ir/dn) against gastrointestinal cancers, including stomach, colon, and pancreas. IGF-Ir/dn suppresses tumorigenicity both in vitro and in vivo and increases stressor-induced apoptosis. IGF-Ir/dn expression upregulates chemotherapy-induced apoptosis and these combination therapies with chemotherapy are very effective against tumors in mice. Some drugs blocking IGF-Ir function are now entering clinical trial, thus IGF-Ir might be a candidate for a therapeutic target in several gastrointestinal malignancies. [source]

Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssion

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004
Edward R. Bastow
Introduction Recent evidence suggests that hyaluronan (HA) facilitates the mechano-dependent joint cavity-forming process through the elaboration and retention of a HA-rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity. Materials and methods Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell-coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA-) or dominant negative (DN-) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059-treated cells were monitored by immunoblotting and medium HA concentrations by ELISA. Results AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA-pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059-treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN-MEK diminished, while CA-MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN-MEK exhibited a significant reduction in labelling of free HA on the cell surface. Discussion These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface-associated HA (ii) PD98059-induced blockade of ERK activation restricts medium HA release and (iii) ERK-mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA-rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line-related differentiation. [source]

Jun N-terminal kinase pathway enhances signaling of monocytic differentiation of human leukemia cells induced by 1,25-dihydroxyvitamin D3

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2003
Qing Wang
Abstract Recent studies revealed that the MEK/ERK module of the mitogen-activated protein kinase (MAPK) signaling cascades is up-regulated in the early stages of 1,,25-dihydroxyvitamin D3 (1,25D3)-induced monocytic differentiation of human leukemia cells HL60. In the present study, we investigated whether another MAPK module, the JNK pathway, also participates in this form of differentiation. We found that the dependence on the concentration of the inducer, the vitamin-hormone 1,25D3, in two types of human leukemia cells, HL60 and U937, and the kinetics of monocytic differentiation in HL60 cells, parallel the degree of the activation of the JNK pathway. A blockade of JNK signaling by a stable expression of dominant negative (dn) JNK1 mutant in U937 cells resulted in reduced c-jun phosphorylation, and the differentiation of these cells was markedly decreased. Similarly, inhibition of JNK1 and JNK2 activities by the selective inhibitor SP600125 led to both dose-dependent reduction of c-jun and ATF-2 phosphorylation, and of the differentiation of HL60 cells. In addition, we found that JNK activity is essential for the AP-1 DNA binding induced by 1,25D3 in HL60 and U937 cells. The results indicate that in cultured human leukemia cells, the JNK pathway participates in the induction of monocytic differentiation by 1,25D3, probably by activating the AP-1 transcription factor. © 2003 Wiley-Liss, Inc. [source]

PI3K/AKT regulates aggrecan gene expression by modulating Sox9 expression and activity in nucleus pulposus cells of the intervertebral disc

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009
Chin-Chang Cheng
The goal of the investigation was to test the hypothesis that the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway regulates the expression of the major extracellular matrix component of the intervertebral disc, aggrecan, in nucleus pulposus cells. Primary rat nucleus pulposus cells were treated with PI3K inhibitor to measure changes in gene and protein expression. In addition, cells were transfected with various luciferase reporter plasmids to investigate mechanisms of regulation of aggrecan gene expression. We found that treatment of nucleus pulposus cells with a PI3K inhibitor, LY294002 resulted in decreased expression of aggrecan and a reduction in deposition of sulfated glycosaminoglycans. Moreover, pharmacological suppression or co-expression of dominant negative (DN)-PI3K or DN-AKT resulted in downregulation of aggrecan promoter activity. Expression of constitutively active (CA)-PI3K significantly induced aggrecan promoter activity. We observed that PI3K maintained Sox9 gene expression and activity: inhibition of PI3K/AKT resulted in decreased Sox9 expression, lowered promoter activity, and mediated a reduction in Sox9 transcriptional activity. PI3K effects were independent of phosphorylation status of C-terminus transactivation domain (TAD) of Sox9. Finally, we noted that in nucleus pulposus cells, PI3K signaling controlled transactivation of p300 (p300-TAD activity), an important transcriptional co-activator of Sox9. Results of these studies demonstrate for the first time that PI3K/AKT signaling controls aggrecan gene expression, in part by modulating Sox9 expression and activity in cells of the nucleus pulposus. J. Cell. Physiol. 221: 668,676, 2009. © 2009 Wiley-Liss, Inc. [source]

Structural alterations in a type IV pilus subunit protein result in concurrent defects in multicellular behaviour and adherence to host tissue

MOLECULAR MICROBIOLOGY, Issue 2 2001
Hae-Sun Moon Park
The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial,substratum and bacterial,bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell,cell interactions to colonization. [source]

Proteomic profiling of tumor cells after induction of telomere dysfunction

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2009
Stefan Zimmermann Dr.
Abstract Cell division in the absence of telomerase causes progressive telomere shortening which ultimately leads to telomere dysfunction and initiation of genome instability. In order to identify factors related to loss of telomere function, the effects of telomerase inhibition on the proteome of five tumor cell lines were followed by SELDI-TOF-MS. Five differentially expressed protein peaks (p<0.01) were found in a total of 60 clones of five cell lines representing four tissues (lung, breast, prostate, and colon) in which telomerase was inhibited by retroviral overexpression of a dominant negative (DN) mutant of human telomerase reverse transcriptase (hTERT). Among these, a 11.3,kDa peak diminished in DN-hTERT clones was identified as histone H4 by nanoflow-HPLC-MS/MS. Immunoblot analysis not only confirmed the decline of histone H4, but also of other core histone proteins including histone H3. Furthermore, upregulation of several cytokeratins was found to be associated with telomere attrition. In conclusion, loss of telomere function is associated with alterations in the proteome which may represent novel biomarkers for the detection of replicative senescence. [source]

A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function

THE JOURNAL OF PHYSIOLOGY, Issue 4 2010
Dai-Fei Wu
The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the ,-aminobutyric acid type A (GABAA) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT3A receptors, we replaced it with alanine and expressed the 5-HT3A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT3A(C312A) mutant, when co-expressed with the wild-type 5-HT3A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT3A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT3A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor , and , subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor ,7 and GABAA, subunits) supported robust function. Thus, 5-HT3A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating. [source]

Gene Transfer of TRPC6DN (Dominant Negative) Restores Erectile Function in Diabetic Rats

Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma,

Refined Geographic Distribution of the Oriental ALDH2*504Lys (nee 487Lys) Variant

ANNALS OF HUMAN GENETICS, Issue 3 2009
Hui Li
Summary Mitochondrial aldehyde dehydrogenase (ALDH2) is one of the most important enzymes in human alcohol metabolism. The oriental ALDH2*504Lys variant functions as a dominant negative, greatly reducing activity in heterozygotes and abolishing activity in homozygotes. This allele is associated with serious disorders such as alcohol liver disease, late onset Alzheimer disease, colorectal cancer, and esophageal cancer, and is best known for protection against alcoholism. Many hundreds of papers in various languages have been published on this variant, providing allele frequency data for many different populations. To develop a highly refined global geographic distribution of ALDH2*504Lys, we have collected new data on 4,091 individuals from 86 population samples and assembled published data on a total of 80,691 individuals from 366 population samples. The allele is essentially absent in all parts of the world except East Asia. The ALDH2*504Lys allele has its highest frequency in Southeast China, and occurs in most areas of China, Japan, Korea, Mongolia, and Indochina with frequencies gradually declining radially from Southeast China. As the indigenous populations in South China have much lower frequencies than the southern Han migrants from Central China, we conclude that ALDH2*504Lys was carried by Han Chinese as they spread throughout East Asia. Esophageal cancer, with its highest incidence in East Asia, may be associated with ALDH2*504Lys because of a toxic effect of increased acetaldehyde in the tissue where ingested ethanol has its highest concentration. While the distributions of esophageal cancer and ALDH2*504Lys do not precisely correlate, that does not disprove the hypothesis. In general the study of fine scale geographic distributions of ALDH2*504Lys and diseases may help in understanding the multiple relationships among genes, diseases, environments, and cultures. [source]

Alternative infectious entry pathways for dengue virus serotypes into mammalian cells

CELLULAR MICROBIOLOGY, Issue 10 2009
Eliana G. Acosta
Summary The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-,-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells. [source]