Expression and functional role of CRIPTO-1 in cutaneous melanoma.

BACKGROUND: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. METHODS: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. RESULTS: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. CONCLUSION: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells.

A gene encoding a protein related to the serpin family of protease inhibitors was identified as a candidate tumor suppressor gene that may play a role in human breast cancer. The gene product, called maspin, is expressed in normal mammary epithelial cells but not in most mammary carcinoma cell lines. Transfection of MDA-MB-435 mammary carcinoma cells with the maspin gene did not alter the cells' growth properties in vitro, but reduced the cells' ability to induce tumors and metastasize in nude mice and to invade through a basement membrane matrix in vitro. Analysis of human breast cancer specimens revealed that loss of maspin expression occurred most frequently in advanced cancers. These results support the hypothesis that maspin functions as a tumor suppressor.

Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential.

BACKGROUND: Several protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas. PURPOSE: Our purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior. METHODS: We evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis. RESULTS: In A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblotting, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070-10 and C1070-14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity. CONCLUSION: These findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines.

Suppression of prostate cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection.

Our previous studies have demonstrated the heterogeneous expression of E-cadherin in a Dunning rat prostate tumor model. From this model, cloned E-cadherin-negative cells exhibited enhanced invasive and metastatic potential when compared with E-cadherin-positive cells. In this report, we examined the invasion suppressor function of E-cadherin in these prostate tumor cell clones. The E-cadherin gene was stably transfected into E-cadherin-negative Dunning clones. E-cadherin transfection resulted in the up-regulation of the three major catenins (alpha-, beta-, and gamma-catenin) and enhanced Ca2+-dependent cellular cohesiveness. Morphological analyses of E-cadherin transfectants revealed a reversion from a fibroblastic, motile phenotype to a more stationary epithelial phenotype. Matrix metalloproteinase 2, an important marker associated with invasive and metastatic potential, was reduced in all six stable transfected lines. A concomitant decrease in cellular invasiveness was observed, as assessed in vitro by the ability of the transfected cells to invade biological matrices. These results lend further support to the hypothesis that in this experimental system, E-cadherin plays a central role in reducing the cellular invasiveness of prostatic adenocarcinoma, due in part to the down-regulation of matrix metalloproteinase 2 activity. Moreover, the data shed additional light on the possible mechanisms involved in E-cadherin-dependent modulation of invasion.

Application of the National Cancer Institute international criteria for determination of microsatellite instability in ovarian cancer.

Recently, the National Cancer Institute (NCI) established criteria for determination of microsatellite instability (MSI) in colorectal tumors. Although the best panel of markers for ovarian tumors is not known, we evaluated epithelial ovarian cancers for MSI based on the NCI recommendations. One hundred and nine ovarian tumors were analyzed for MSI by gel analysis of paired germ-line and tumor DNA. PCR amplification was performed using the panel of five microsatellite markers recommended by the NCI (BAT25, BAT26, D5S346, D2S123, and D17S250) and nine additional markers picked based on their genomic location (NME1, D10S197, D11S904, D13S175, DXS981, DXS6800, DXS6807, AR, and D3S1611). Tumors were characterized on the basis of: high-frequency MSI (MSI-H) if two or more of the five NCI markers showed instability or there was instability at 30% or more of all markers tested; or low-frequency MSI (MSI-L) if only one of the five NCI markers showed instability or <30% of all of the markers. All of the other tumors were considered microsatellite stable. On the basis of the NCI markers, 12 (11%) tumors demonstrated MSI-H, and 8 (7%) additional tumors had MSI-L. When all of the 14 markers were considered together, 13 (12%) tumors demonstrated MSI-H (based on 30% or more unstable loci), and 26 (24%) tumors had MSI-L. A single tumor identified to have MSI-H based upon all of the markers tested would have been classified as MSI-L based upon the NCI markers alone. Inclusion of an additional dinucleotide marker (NME1) to the NCI panel allowed detection of all of the tumors with MSI-H using only six markers. MSI-H occurs in approximately 12% of invasive ovarian tumors. For optimal detection of microsatellite instability in ovarian cancer, an additional marker (NME1) may be required, along with the five recommended by the NCI.

The 72 kDa type IV collagenase is modulated via differential expression of alpha v beta 3 and alpha 5 beta 1 integrins during human melanoma cell invasion.

We have recently reported that concomitant with an increase in invasiveness, there is an increase in the expression and secretion of the matrix-degrading 72 kDa gelatinase A/type IV collagenase (MMP-2) in a moderately invasive human melanoma cell line (A375M) upon perturbation of the alpha v beta 3 classic vitronectin receptor. In the present study, we have extended these observations to include a highly invasive and metastatic melanoma cell line (C8161) which expresses a comparable amount of the alpha 5 beta 1 integrin (classic fibronectin receptor), but very little alpha v beta 3 integrin on its surface. When perturbed with an anti-alpha 5 beta 1 antibody, C8161 cells are 89% more invasive in vitro, and express and secrete increased levels of the gelatinase A. These changes were not elicited using antibodies to the alpha v beta 3 integrin. In addition, a 73% increase in invasion of C8161 cells through a fibronectin-enhanced matrix occurred, which could be abrogated by neutralizing antibodies to gelatinase A. Furthermore, we attempted to transiently mimic the invasive phenotype of the C8161 cells by diminishing the alpha v beta 3 integrin from the A375M cell surface through fluorescence-activated cell sorting selection or deoxynojirimycin treatment, and found these cells to be 30-50% more invasive than the parental population. These data suggest that alternative modulation and signaling events could be involved in melanoma tumor cell invasion as a result of the differential expression of integrins, and strictly cataloging the presence of these integrins is but an initial step in the analysis of their functional activity.

maspin suppresses the invasive phenotype of human breast carcinoma.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

Inhibition by retinoic acid of type IV collagenolysis and invasion through reconstituted basement membrane by metastatic rat mammary adenocarcinoma cells.

The activity of type IV collagenase, which enables tumor cells to degrade collagen type IV found in the subendothelial basement membrane, has been correlated with the metastatic potential in several tumor types, including the rat 13762NF mammary adenocarcinoma cell line and its clones. In this study, we examined whether all-trans-retinoic acid (all-trans-RA) and other retinoids, which exhibit antitumor activity in vitro and in vivo, affect the collagenolytic activity of metastatic rat 13762NF mammary adenocarcinoma cells. Cells of the highly metastatic lung-colonizing clone MTF7.T35.3, derived from the 13762NF cell line, were treated for 3 days with 0.1, 1, or 10 microM all-trans-RA, harvested, and seeded on [3H]proline-labeled extracellular matrix deposited by cultured rat lung endothelial cells or on a film of purified [3H]proline-labeled type IV collagen. The amount of radioactivity released into the medium during the subsequent 24 to 72 h was measured, and it was found that all-trans-RA treatment inhibited degradation of extracellular matrix and type IV collagen by 50 to 60%. This effect was observed whether the cells had been treated with all-trans-RA in serum-free medium or in medium supplemented with heat-inactivated or acid-treated fetal bovine serum. The growth of the cells was not inhibited under these conditions, except after treatment with 10 microM all-trans-RA in serum-free medium. The reduction in collagenolytic activity was observed in viable cells as well as in conditioned medium. A 24-h exposure of cells to all-trans-RA was sufficient to cause a 30% decrease in the collagenolytic activity, and this inhibitory effect was reversible. The direct addition of all-trans-RA to conditioned medium had no effect on secreted collagenase activity. The apparent molecular weights of the collagenolytic enzymes were determined by electrophoresis of cell extracts and concentrated conditioned medium in type IV collagen-embedded polyacrylamide gels followed by renaturation and activation of the enzymes within the gels. Two major type IV collagenolytic metalloproteinases exhibiting molecular weights of 64,000 and 88,000, respectively, were detected by this method. These two enzymes were also found to have specificity for gelatin. The Mr 64,000 enzyme could be extracted from viable cells (presumably from the cell membrane) by 2% 1-butanol. Treatment with all-trans-RA decreased the level of these enzymes in the cellular, cell membrane, and conditioned medium compartments.(ABSTRACT TRUNCATED AT 400 WORDS)

v-jun oncogene suppresses both phorbol ester-induced cell invasion and stromelysin gene expression in a mouse papilloma cell line.

The viral jun (v-jun) oncogene encodes a transcription factor that can participate in the transactivation of genes through the AP-1 complex. Evidence indicates that the ability of v-jun to transform cells and stimulate transcription depends on the cell type. We have asked whether expression of the v-jun gene in benign tumor forming mouse keratinocytes that already express an activated c-rasHa oncogene would cause malignant progression. Our results showed that the v-jun transfection did not result in malignant progression; instead, we made the unexpected observation that the ability of these cells to invade reconstituted basement membrane matrix (in vitro) in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was suppressed. This phenomenon could, in part, be explained by the suppression of the induction by phorbol ester of expression of the metalloproteinase, stromelysin (transin). Of interest was the finding that 12-O-tetradecanoylphorbol-13-acetate induction of other cellular genes known to be regulated by AP-1 was not inhibited in the benign tumor cells expressing v-jun.

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.

Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Retinoic acid inhibition of human melanoma cell invasion through a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor.

Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.

Maspin: a tumor suppressing serpin.

Maspin, a serpin found in mammary epithelial cells, has been shown to have tumor suppressor activity. The gene is expressed in normal human mammary epithelial cells but down-regulated in invasive breast carcinomas. Similar patterns of expression at the RNA and protein levels are seen by Northern analysis with cells grown in culture and by immunostaining of tissues. Biological assays of invasion by tumor cells through matrigel membranes and of motility have shown that recombinant maspin inhibits both processes, and that its inhibitory action is totally lost by a single cleavage at the reaction center. Tumor transfectants expressing maspin are inhibited in growth and invasion in nude mice. Maspin is located in the cell membrane and extracellular matrix, and does not behave as a classical inhibitory serpin against any known target protease. Its mode of action is presently unknown.

Association between keratin and vimentin expression, malignant phenotype, and survival in postmenopausal breast cancer patients.

Pathology observational reports and experimental data suggest that keratin and vimentin intermediate filament (IF) coexpression in breast cancer confers a more aggressive "interconverted" phenotype, expressing both epithelial and mesenchymal markers. In this study, we extended previous observations by measuring the expression of keratin and vimentin, in relation to other selected biomarkers of disease progression, in postmenopausal women with breast cancer. Using immunohistochemical analysis of 54 archival, formalin-fixed, paraffin-embedded invasive breast cancers from a well-defined cohort, we examined relative IF (keratin and vimentin) expression in a semiquantitative fashion and compared these results with other biological markers and survival. By univariate analysis, we found that vimentin expression was inversely associated with keratin expression alone (P = 0.0089) and directly related to histological grade (P = 0.017), nuclear grade (P = 0.027), Ki67 growth fraction (P = 0.024), and epidermal growth factor receptor immunostaining (P = 0.019). The relative expression of keratin and vimentin in approximately similar amounts characterized tumors with the poorest prognosis, as compared with keratin-high/vimentin-negative or keratin-low/vimentin-positive tumors. These latter two groups demonstrated similar Kaplan-Meier survival curves; the former group (keratin and vimentin in approximately similar amounts) demonstrated a poorer survival, with a hazard ratio of 2.1 (95% confidence interval, 0.5-9.6). These data suggest that relative keratin and vimentin IF expression is more indicative of prognosis and tumor phenotype than either IF marker detected independently.

Effects of antioxidant enzyme overexpression on the invasive phenotype of hamster cheek pouch carcinoma cells.

To examine the role of reactive oxygen species on the invasive phenotype of cancer cells, we overexpressed manganese- and copper-zinc-containing superoxide dismutases (MnSOD, CuZnSOD) and catalase (Cat) in hamster cheek pouch carcinoma (HCPC-1) cells in vitro using adenoviral vector-mediated gene transfer. Hamster cheek pouch carcinoma cells were transduced with these adenoviral vector constructs alone, or in combination, at concentrations [i.e., multiplicity of infectivity (MOI)] of 100 MOI each. The Escherichia coli beta-galactosidase reporter construct was used as a control virus. Protein expression was examined by Western blot analysis and enzymatic activities were measured using spectrophotometry. To observe the effects of transgene overexpression on in vitro tumor cell invasion, we used the membrane invasion culture system, an accurate and reliable method for examining tumor cell invasion, in vitro. This assay measures the ability of tumor cells to invade a basement membrane matrix consisting of type IV collagen, laminin, and gelatin. MnSOD overexpression resulted in a 50% increase in HCPC-1 cell invasiveness (p < .001); co-overexpression of MnSOD with Cat partially inhibited this effect (p < .05). Moreover, co-overexpression of both SODs resulted in a significant increase in invasiveness compared with the parental HCPC-1 cells (p < .05). These changes could not be correlated with the 72 kDa collagenase IV or stromolysin activities using zymography, or the downregulation of the adhesion molecules E-cadherin or the alpha4 subunit of the alpha4beta1 integrin. These results suggest that hydrogen peroxide may play a role in the process of tumor cell invasion, but that the process does not rely on changes in matrix metalloproteinase activity in the cells, or the expression of cell adhesion molecules.

In vitro quantification of melanoma tumor cell invasion.

In order to quantify the invasiveness of melanoma tumor cells in vitro, a modification of the amniotic basement membrane (BM) model, described by Liotta et al. (Cancer Letters, 11, 141, 1980), was used in combination with radiolabeled tumor cells. B16-F10 metastatic murine melanoma cells and a derived clone (B16-F10L) were prelabeled with 0.1 muCi/ml of [14C]thymidine for 20-24 h in serum-free medium at 37 degrees C. Following incubation, fetal bovine serum was added to a concentration of 5 per cent, and the cells were allowed to grow to confluency for the next 24-28 h. The labeled cells were seeded onto amniotic membranes situated in Membrane Invasion Culture System (MICS) chambers at a density of 2.5 X 10(4) per well. At various times points, radioactivity of tumor cells that completely traversed the membrane was determined using an under-the-membrane sampling method. The average percent invasion demonstrated by the B16-F10 line was 2.75 per cent, and 3.65 per cent exhibited by the B16-F10L cell line after 48-53 h in vitro. Since it was apparent that some variability in thickness existed among membrane samples, a morphological analysis was performed on five sectors of a three-inch-diameter sample from four different placentae. Differences and similarities in BM thickness within the same sector were noted by this technique and could possibly contribute to some variability observed in tumor cell invasion in this model. Another parameter examined was the proliferation of tumor cells in the upper and lower wells of the MICS chambers. By 48 h, approximately 32.1 per cent of the B16-F10 cell line as well as the clone had replicated in the upper wells associated with the BMs compared with a 32.9 per cent replication in the lower wells, which reaffirmed the viability of the tumor cells under experimental conditions and insured similarly replicating populations of cells. In order to quantify the invasiveness of radiolabeled tumor cells accurately through a biological membranous barrier, the proper concentration of cells must be used, tumor cell heterogeneity should be taken into consideration, the technique of sampling radiolabeled invasive cells should be critically analysed, and thickness of the membranous barrier should all be considered as possible important factors in the quantitative analyses.

Adriamycin-induced inhibition of melanoma cell invasion is correlated with decreases in tumor cell motility and increases in focal contact formation.

Tumor cell adhesion to the extracellular matrix (ECM) is closely linked with tumor cell invasion and metastasis. In this study, we demonstrate that low levels of adriamycin, a widely used anticancer drug, can inhibit the invasion of highly metastatic K1735-M2 mouse melanoma cells in vitro through a reconstituted basement membrane extract. Adriamycin-induced inhibition of melanoma cell invasion occurred at levels of the drug (i.e. 1 ng/ml) that did not inhibit tumor cell growth, suggesting that the observed inhibition in tumor cell invasion was not due to the well-documented ability of adriamycin to interfere with DNA and/or RNA synthesis. Rather, these studies indicated that adriamycin-induced inhibition of melanoma cell invasion was accompanied by a corresponding decrease in the ability of adriamycin-treated tumor cells to migrate in response to several isolated ECM components including fibronectin, laminin and basement membrane (type IV) collagen. The decreased migration of adriamycin-treated tumor cells was not accompanied by a decrease in the adhesion or spreading of the adriamycin-treated cells on substrata coated with these ECM components. Instead, adriamycin-treated cells actually exhibited a slightly increased propensity (compared to untreated control cells) to adhere on fibronectin-, laminin-, and type IV collagen-coated substrata. Additionally, adriamycin treatment caused a dramatic increase in focal contact formation by these melanoma cells, as assessed by fluorescent microscopy of actin and vinculin. In addition to providing a useful model for which to study the molecular and cellular basis for focal contact formation, these results further emphasize the results of several other investigators that have suggested an important role for focal contacts in modulating tumor cell motility, invasion and metastasis.