Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy.

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems.

In 1998 we reported that an L-peptide derived from H1 of c-Myc (Int-H1-S6A,F8A), linked to an internalization sequence from the third a-helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro-inverso (RI-) peptides can assume a 3D conformation very close to their corresponding L-forms and can be recognized by the same monoclonal antibody.

TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis.

The matrix metalloproteinase (MMP) inhibitor TIMP-2 has a high specificity for gelatinase A/MMP-2. An imbalance between gelatinase A and TIMP-2 in favor of enzymatic activity is linked to the degradation of the extracellular matrix (ECM) associated with several physiologic and pathologic events, including angiogenesis, invasion and metastasis. Since TIMPs are secreted molecules, they have the potential to be used for gene therapy of certain tumors.

The condensation of chromatin in apoptotic thymocytes shows a specific structural change.

Chromatin condensation and DNA cleavage at internucleosomal sites have been recognized early as hallmarks of apoptosis, and it has been suggested that extensive DNA chain scission could directly result in the formation of dense chromatin bodies. Here we have shown that no causal relationship exists between DNA degradation and chromatin condensation in glucocorticoid-induced thymocyte apoptosis. The chromatin rearrangement occurred independent of as well as prior to DNA cleavage and involved a specific conformational change at the nucleosome level.

RB1 oncosuppressor gene over-expression inhibits tumor progression and induces melanogenesis in metastatic melanoma cells.

The retinoblastoma gene (RB1) is frequently deleted or mutated in many tumor types and in all cases of retinoblastoma. Apart from its role in regulation of the cell cycle, the RB1 gene product (p110RB1) appears to be involved in control of differentiation. Malignant metastatic cells show many properties of poorly differentiated cells, and are highly invasive in vitro and in vivo.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes.

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice.

The alpha 3 beta 1 integrin is involved in melanoma cell migration and invasion.

The VLA3 (alpha 3 beta 1) integrin receptor recognizes several ligands; however, the function of this integrin is still debated. Expression of VLA3 appears to be increased in malignant melanoma and correlates with the degree of dermal invasiveness. Here we have studied the role the alpha 3 integrin subunit in malignant melanoma cell migration and invasion into extracellular matrices.

Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing.

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5.

Production of angiogenesis inhibitors and stimulators is modulated by cultured growth plate chondrocytes during in vitro differentiation: dependence on extracellular matrix assembly.

Secretion of angiogenesis inhibitors and stimulators is modulated during in vitro differentiation of embryonic chick growth plate chondrocytes. Supernatants from dedifferentiated cells undergoing maturation to hypertrophic chondrocytes in suspension progressively inhibited vascular cell random migration and invasion of basement membrane matrix by endothelial cells. Maximal inhibition was exhibited by conditioned medium from hypertrophic chondrocytes.

Retinoic acid negatively regulates beta 4 integrin expression and suppresses the malignant phenotype in a Lewis lung carcinoma cell line.

Retinoic acid (RA) is a potent inhibitor of the malignant phenotype and of tumour cell growth. We observed that in vitro RA treatment of a highly metastatic lung carcinoma cell line (C87) induced a marked reduction in the amount of the beta 4 integrin subunit. The downregulation of this adhesion molecule was assessed by immunofluorescence, immunoprecipitation, and northern analysis.

Metalloproteinase and TIMP expression by the human breast carcinoma cell line 8701-BC.

It is widely accepted that collagenolytic enzymes are required to facilitate the invasion and spread of tumour cells into host tissues. Immunohistochemical, zymographic and PCR analyses have produced evidence that the recently established human mammary carcinoma cell line, 8701-BC, expresses several metalloproteinases (MMP-1, -2, -9 and -10) and their tissue inhibitors (TIMP-1 and -2). Application of these different techniques has led to several observations, both complementary and dissimilar.

Induction of a malignant phenotype in BALB/c 3t3 cells by 1,1,2,2-tetrachloroethane.

BALB/c 3T3 cells transformed by 1,1,2,2-tetrachloroethane, the most toxic chloroethane, acquired a fully malignant phenotype. They were capable of growing in soft agar, were tumorigenic when injected in athymic mice and produced pulmonary nodules after i.v.

Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes.

Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro reconstituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immunocytochemistry of in situ epidermis and were found to expose only the integrin subunits alpha 3, alpha 6, alpha v and beta 1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha v beta 3, which were immunoprecipitated from both metabolically and surface-labeled cells.

Matrigel promotes retinoblastoma cell growth in vitro and in vivo.

Cells derived from retinoblastomas grow slowly in vitro and only very rarely form tumors in nude mice. Matrigel, a mixture of components normally found in basement membranes, promotes the growth of Y-79 and WERI-Rb1 retinoblastoma (Rb) cells when added to suspension cultures of the 2 Rb cell lines. It also substantially increases cell adhesion in vitro.

Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment.

The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site.

Laminin-induced retinoblastoma cell differentiation: possible involvement of a 100-kDa cell-surface laminin-binding protein.

Gene and protein expression of Y-79 retinoblastoma cells growing on poly(D-lysine) is switched from a photoreceptor-like to a conventional neuron-like pathway by the basement membrane glycoprotein laminin. Unlike other cell systems where laminin influences differentiation, Y-79 cells can neither attach to nor chemotactically respond to laminin. However, laminin increases attachment to poly(D-lysine).

Induction of invasive and experimental metastasis potential in BALB/c 3T3 cells by benzo(a)pyrene transformation.

A clone of BALB/c 3T3 cells (A-31), which is highly resistant to spontaneous in vitro transformation, was treated with the carcinogen benzo(a)pyrene [B(a)P]. This agent was capable of inducing in vitro transformation in the presence of S9 activating system and 6 weeks after treatment large foci were detected. Transformation frequency in solvent control groups was very low.

Tumor cell invasion inhibited by TIMP-2.

The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness.

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells.

Increased invasive, chemotactic and locomotive abilities of c-Ha-ras-transformed human breast epithelial cells.

Transfection of the immortalized human breast epithelial cells MCF-10A with the mutated ras oncogene resulted in cell transformation (MCF-10A-neoT). Since the transformed state is usually associated with enhanced migratory activity, increased capability to invade basement membranes and to grow in a three-dimensional basement membrane gel (growth in matrigel), we compared these properties in MCF-10A-neoT cells with those of MCF-10A cells transfected with either the neomycin resistance gene alone (MCF-10A-neo cells) or with the normal ras proto-oncogene (MCF-10A-neoN cells). MCF-10A-neoT cells exhibited enhanced migratory activity, as assessed by chemotaxis and chemokinesis assays.

Invasive activity, spreading on and chemotactic response to laminin are properties of high but not low metastatic mouse osteosarcoma cells.

We have examined the interactions of low (Os43 and OS48) and high (Os50/K8 and Os50/K12) metastatic cell lines derived from osteosarcomas (Os) of the Balb/c mouse with fibronectin (FN) and laminin (LN). All of these cell lines formed osteogenic tumors when transplanted subcutaneously into syngeneic mice. Os43 and Os48 cells gave rise to few metastases while the Os50/K8 and Os50/K12 cells were highly metastatic.

[Use of reconstituted basal membranes for the study of invasion of human tumor cells: current status and future prospects].

Tumor metastasis is the major cause of death of oncology patients. One of the characteristic properties acquired by the metastatic cell is the ability to cross basement membranes. These are compartments of extracellular matrix composed largely by collagen type IV, laminin and a heparan sulphate proteoglycan.

Invasiveness and chemotactic activity of oncogene transformed NIH/3T3 cells.

The role of oncogenes in the acquisition of invasive and metastatic capabilities is controversial. Interactions with basement membranes are critical in the process of tumor invasion and metastasis. We compared the ability of 3T3 cells transformed by oncogenes involved in various stages of signal transduction to invade a reconstituted basement membrane in vitro and to grow in a three dimensional basement membrane gel (matrigel).