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. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

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. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as "emergency" receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor.

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.

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. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).

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. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.

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. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the "critical" cysteine residue, were added at 30 microM concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier; control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.

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. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

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. We have transfected the human RB1 cDNA in an expression vector under the control of the beta-actin promoter into B16F10 murine melanoma cells. These cells highly overexpress RB1 mRNA and the p110RB1 product, show reduced growth rate and increased melanogenesis in vitro. Vector control transfectants showed no alteration of invasiveness. The p110RB1 over-expressing cells also had a reduced capacity to migrate and invade through an artificial basement membrane, key characteristics of metastatic cells. When injected into nude mice, the p110RB1 over-expressing cells showed reduced tumor growth and reduced metastatic potential. The few metastasis observed were predominantly melanotic. These data indicate that RB1 gene expression is involved in melanoma cell differentiation and plays a role in downregulation of migration, invasion and metastatic potential of these cells.

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. The same medium also repressed vascular cell migration induced by highly angiogenic Kaposi's sarcoma cell supernatants and prevented formation of an anastomosed network of tube-like structures by endothelial cells plated on matrigel. On the contrary, when the suspension culture of hypertrophic chondrocytes was supplemented with ascorbic acid, a condition leading to the formation of a mineralized tissue similar to calcified cartilage, a dramatic switch to production of angiogenic activity was observed. Medium conditioned by osteoblast-like cells derived from hypertrophic chondrocytes also induced vascular cell migration and invasion of basement membrane matrix. The presence of angiogenic activity in the conditioned medium was assessed also by an in vivo assay in mice using reconstituted basement membrane associated with heparin. Therefore, interactions of chondrocytes with their extracellular matrix are an absolute requirement for the expression of angiogenic activities by hypertrophic chondrocytes at late developmental stages.

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. In order to investigate the effects of RA on the malignant phenotype in C87 cells we performed morphological and functional analysis after RA treatment. We found that RA was able to produce marked changes in C87 cell shape, increasing the number of flat cells (90% of the total cell population), and significantly inhibiting the malignant and invasive phenotype of C87 cells. RA treatment suppressed their clonogenic potential in soft agar (control, 20 +/- 5; RA, 0), and strongly reduced their chemotactic and chemoinvasive capacity (chemotaxis: control, 231 +/- 5; RA, 28 +/- 0; chemoinvasion: control, 132 +/- 11; RA = 2 +/- 1). FACS analysis and cell count, however, indicated that RA reduced the growth of C87 cells only partially. After 72 h of treatment we observed only a 10% reduction in the S phase fraction of the cell population. Finally, the reduced lung colony-forming ability, observed after i.v. injection of RA-treated cells (lung foci/animal: RA-treated cells, 1 +/- 0.1; untreated, 8.5 +/- 0.8), further supports the conclusion that in this murine lung carcinoma cell line a marked reduction in the expression of the beta 4 integrin subunit is associated with a marked inhibition of the malignant phenotype.

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. injection. Untreated BALB/c 3T3 cells were weakly tumorigenic (3/9 animals developed tumors after a longer time) and induced rare pulmonary nodules in a low percentage of animals. Transformed cells were also invasive in the chemoinvasion assay. Moreover, they grew in a gel of reconstituted basement membrane (matrigel), penetrating the gel and assuming a branching morphology that has been associated to the malignant phenotype. By contrast, spontaneous transformants isolated from solvent controls were poorly invasive in these assays. These results show that 1,1,2,2-tetrachloroethane could play a role in the late steps of multistage carcinogenesis.

Interferons inhibit chemotaxis of transformed cells and their invasion of a reconstituted basement membrane.

Tumor cell migration and invasion are critical steps in the complex process of metastasis formation. It has been demonstrated that interferons (IFNs) inhibit the motility of human fibroblasts. In the present investigation we tested the effect of human leukocyte IFN and murine fibroblast IFN on the chemotactic migration of transformed and tumor-derived cells towards fibroblast conditioned medium. We were able to show that IFNs preferentially inhibit the chemotaxis of transformed and tumor-derived cell lines when compared to control fibroblasts. Inhibition was dose-dependent and most tumor cell strains were sensitive to concentrations of IFNs 10- to 100-fold lower than fibroblast cultures. Furthermore, leukocyte interferon was able to inhibit the invasion of transformed 3T3 fibroblasts through a gel of reconstituted basement membrane (Matrigel). These effects could be related to the antineoplastic activity of interferon.

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). Cell lines transformed by various oncogenes and oncoviruses: v-sis (a growth factor), v-erb-B (a truncated EGF receptor), Moloney sarcoma virus (v-mos: a protein kinase homologue), mutated c-ras oncogenes (G protein homologues), FBJ virus (v-fos: a nuclear protein) were investigated. All transformed cell lines were able to invade in the chemoinvasion assay, where a layer of matrigel is coated onto chemotaxis filters. FBJ/3T3 were the least invasive and SSV/3T3 the most invasive. Control 3T3 cells could not cross the matrigel barrier. All transformed cells grew on matrigel forming invasive, branching colonies, whereas control 3T3 were unable to grow in matrigel. Cells transfected with the v-erb-B gene grew as multilayers inside matrigel. Invasiveness and growth on matrigel were accompanied by a high chemotactic response to laminin (LN) in all transformed lines. These results suggest that invasion and growth on matrigel, together with migration to LN, are induced by a large spectrum of oncogenes. When 3T3 cells were transfected with v-sis oncogene under the transcriptional control of the metallothionein (MMT) promoter and exposed to Zn++, their in vitro invasiveness was specifically increased by around 3 fold. These findings provide further evidence supporting a direct role of the v-sis oncogene in the invasive phenotype.

High chemotactic motility and growth in hard agar of a variant of RSV-transformed fibroblasts are lost in late passages.

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6% agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.

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. Beta 1 integrins were diffused on the adhesion surface, while alpha v beta 3 was clustered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific beta 1 integrins; (ii) spreading is mainly modulated by alpha v beta 3; (iii) the beta 1 and beta 3 heterodimers cooperate in regulating migration. The in vitro expression of two integrins (alpha v beta 3 and alpha 5 beta 1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a normally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of alpha 3 beta 1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.

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. and increased capability to invade the basement membrane. These cells also formed large colonies in matrigel. MCF-10A-neo and MCF-10A-neoN cells, on the other hand, showed only marginal migratory, invasive and semisolid medium growth properties. These results indicate that the mutated ras oncogene induces in human breast epithelial cells phenotypic characteristics of malignant transformation.

Establishment and characterization of osteogenic cell lines from a spontaneous murine osteosarcoma.

Five clonal cell lines were established from a spontaneous BALB/c mouse osteosarcoma, and characterized. Four of these lines showed some similarities in morphology, in vitro growth properties, production of collagenous and noncollagenous extracellular matrix proteins and osteogenic differentiation. The cells formed colonies with characteristic differences in size and morphology in soft agar, and osteogenic sarcomas and metastases in syngeneic mice after transplantation. Ultrastructurally, cells in the transplant tumours showed marked osteogenic features. There were no osteoclast-like cells. The fifth cell line had somewhat different characteristics. All five lines expressed infectious endogenous murine leukemia viruses. Increased c-myc protoon-cogene expression was found in one cell line and c-fos expression at different levels in all lines. There was only very low expression of c-Ha-ras and no expression of c-Ki-ras and c-sis. DNA analysis showed the presence of newly acquired proviral genomes integrated at different sites in the cellular DNA. The results show that distinct osteogenic neoplastic subclones can be obtained from a primary mouse osteosarcoma. Although the clones exhibited an appreciable morphological, functional, and molecular diversity they retained the basic pathogenic properties of the tumour from which they were derived.

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. Whereas PCR analysis showed that mRNA was detected for each of the proteins, the immunolocalization study demonstrated that MMP-1, MMP-2, MMP-9 and TIMP-1 production was restricted to only a proportion of the tumour cells, with no evidence of MMP-3 or TIMP-2 synthesis. Such observations suggested phenotypic heterogeneity within the cell line, which was further examined by use of the tumour cell clones BC-3A and BC-61 derived from the parental 8701-BC line. Comparative studies using zymography and PCR analysis demonstrated differences in MMP-2 and MMP-10 expression between the 3 cultures. The data indicate that the 8701-BC cell line retains an inherent capacity for metalloproteinase and TIMP expression, with the production of both interstitial collagenase (MMP-1) and the 2 basement-membrane-degrading enzymes (MMP-2 and MMP-9) representing an aggressive collagenolytic phenotype. The concomitant production of TIMP-1 by these cell cultures, and the apparent phenotypic heterogeneity displayed by these lines, suggest that metalloproteinase dysregulation may represent an important feature of clonal heterogeneity. Although the 8701-BC and BC-61 cells were much more invasive than those of the BC-3A clone, as judged by the penetration of "Matrigel", it has not yet been possible to relate this invasive potential to the metalloproteinase and TIMP profiles reported here for each cell line.