Diabetogenic glucose and insulin concentrations modulate transcriptome and protein levels involved in tumour cell migration, adhesion and proliferation.

BACKGROUND: During the last decade, epidemiological studies uncovered the tremendous impact of metabolic syndrome/diabetes mellitus type 2 (DM T2) as risk factors of the progression of cancer. Therefore, we studied the impact of diabetogenic glucose and insulin concentrations on the activities of tumour cells, because little is known about how high glucose and insulin levels are influencing gene activities causing changes in the signal cascade activities with respect to kinases involved in the proliferation and migration of cancer cells. METHODS: To address this question we analysed the activity of more than 400 gene signatures related to (i) cell cycle, (ii) cell movement as well as (iii) signal transduction. We examined transcriptomes of kinases (PKCα, PI3K), cadherins (E-, N- VE-), integrins and cyclins by comparing physiological (5.5 mM) vs diabetogenic (11 mM) glucose concentrations (without and with insulin). RESULTS: Proliferation assays revealed that high levels of glucose (11 mM) and insulin (100 ng ml(-1)) did promote the proliferation of the tumour cell lines HT29, SW480, MCF-7, MDA MB468, PC3 and T24. Using a 3D-migration assay, we have shown that high glucose concentrations caused increased motility rates of the tumour cells. The increase in migratory activity at high glucose and insulin concentrations was mediated by an activation of PI3K, PKCα and MLCK, as figured out by the pharmacological inhibitors wortmannin, Go6976 and ML-7. CONCLUSION: We present molecular and functional data, which could help to understand how hyperglycaemia and hyperinsulinemia might trigger tumour cell proliferation and motility in patients, too.

Alpha- and beta-adrenergic receptor (AR) protein expression is associated with poor clinical outcome in breast cancer: an immunohistochemical study.

Breast cancer mortality is frequently associated with metastatic disease. Metastasis models have shown adrenoceptor (AR) stimulation induces cell migration which is inhibited by adrenoceptor antagonist drugs. We investigated adrenoceptor protein expression in clinical breast tumours and its association with disease progression and prognosis. Immunohistochemistry on tissue microarrays was used to characterise α1b, α2c and ß(2)2 adrenoceptor protein expression in operable breast tumours. Associations with tumour-relevant biological markers and clinical outcome were statistically assessed. Strong α1b expression occurred in large high grade (P < 0.0001), HER2+ (P < 0.0001) or basal-like (CK5/6, P = 0.0005; CK14, P = 0.0001; EGFR, P = 0.003) cancers, showing increased proliferation (Mib1, P = 0.002), decreased apoptosis (Bcl2, P < 0.0001) and poor NPI membership (P = 0.001). α1b expression correlated with poor cancer-specific survival (LR = 7.628, P = 0.022) and tumour recurrence (LR = 6.128, P = 0.047). Strong α2c was over-expressed in high grade (P = 0.007), HER3+ (P = 0.002) and HER4+ (P < 0.0001) cancers with borderline increase in EGFR, p53 and MIB1 proteins, and inverse association with hormonal (PgR, P = 0.002) phenotype. In contrast, strong ß(2) expression occurred in small-size, luminal-like (ER+, P < 0.001) tumours of low grade (P < 0.001) and lymph node stage (P = 0.027) that showed poor prognosis when hormonal treatment was withheld. Adrenoceptors were not found to be independent predictors of clinical outcome. Alpha1b and α2c AR is over-expressed in basal-like breast tumours of poor prognosis. Strong ß(2) adrenoceptor expression is seen in patients with a luminal (ER+) tumour phenotype and good prognosis, due to benefits derived from hormonal therapy. These findings suggest a possible role for targeted therapy using adrenoceptor antagonists.

High PKC alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells.

The protein kinase C (PKC) is a family of serine/threonine kinases that are key regulatory enzymes involved in growth, differentiation, cytoskeletal reorganization, tumor promotion, and migration. We investigated the functional involvement of PKC isotypes and of E-cadherin in the regulation of the locomotion of six human colon-adenocarcinoma cell lines. The different levels of the PKC alpha and the E-cadherin expression have predictable implications in the spontaneous locomotory activity. With the use of PKC alpha--specific inhibitors (safingol, Go6976) as well as the PKC delta--specific inhibitor rottlerin, we showed that only PKC alpha plays a major role in the regulation of tumor cell migration. The results were verified by knocking out the translation of PKC isozymes with the use of an antisense oligonucleotide strategy. After stimulation with phorbol ester we observed a translocation and a colocalization of the activated PKC alpha at the plasma membrane to the surrounding extracellular matrix. Furthermore, we investigated the functional involvement of E-cadherin in the locomotion with the use of a blocking antibody. A high level of PKC alpha expression together with a low E-cadherin expression was strongly related to a high migratory activity of the colon carcinoma cells. This correlation was independent of the differentiation grade of the tumor cell lines.

Functional hierarchy of simultaneously expressed adhesion receptors: integrin alpha2beta1 but not CD44 mediates MV3 melanoma cell migration and matrix reorganization within three-dimensional hyaluronan-containing collagen matrices.

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Norepinephrine-induced migration of SW 480 colon carcinoma cells is inhibited by beta-blockers.

Beta-adrenoceptors are highly expressed on SW 480 colon carcinoma cells as was assessed by flow cytometry. We investigated the influence of norepinephrine on the migration of these cells using time-lapse videomicroscopy. Norepinephrine-treatment increased the locomotor activity within the population from 25% spontaneously locomoting cells to 65% locomoting cells. The beta1/2-blocker propranolol but not the beta1-blocker atenolol inhibited this increase. The intracellular signaling solely of norepinephrine-induced locomotion involved protein tyrosine kinase activity, whereas both spontaneous and norepinephrine-induced migration were reduced by inhibiting phospholipase Cgamma and protein kinase Calpha activity. In summary, norepinephrine-induced locomotion of SW 480 cells is beta2-adrenoceptor mediated and distinct from spontaneous locomotion concerning the PTK involvement.

Isolation of prostate-derived single cells and cell clusters from human peripheral blood.

The detection of blood-borne prostate cancer (PCA) cells may help with clinical staging and the further understanding of PCA metastases. We discovered prostate-specific antigen (PSA)-positive stained but not PSA mRNA-expressing blood cells by means of cell sorting and PSA reverse transcription-PCR in patients. Therefore, we developed a cytokeratin immunomagnetic method to isolate PSA-positive epithelial cells from the circulating blood of PCA patients. We obtained blood-borne single cells from 6 of 10 PCA patients and clustered cells from 8 of 10 PCA patients. Patients with benign prostate hyperplasia tested negative for cell clusters. The reported isolation method yielded prostate-derived cells or clusters of them from PCA-diagnosed patients.

Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44.

The three-step model of cell migration consisting of protrusion of a leading lamella, attachment to the substrate, and contraction of the cell body is well established for fibroblasts migrating across planar surfaces. However, it is not resolved to what extent the migration of cancer cells in a 3-dimensional tissue environment follows similar principles. Here, we present evidence that the migration of highly invasive MV3 melanoma cells in 3-dimensional collagen matrices follows the three-step concept of migration but also results in characteristic reorganization of the extracellular matrix. After incorporation in the lattice, MV3 cells spontaneously developed a slow type of migration (mean velocity, 0.19 microm/min), leading to alignment of collagen fibers at attachment sites, as detected from unfixed and fixed samples by confocal reflection contrast in combination with immunofluorescence staining. In the process of migration, the formation of focal clusters or stripes of alpha2 and beta1 integrins colocalized with binding sites to collagen fibrils at the leading as well as the trailing edge. In contrast, CD44 was nonclustered and redistributed toward the rear end of the cell. At detachment sites, dynamic fiber traction, localized fiber disruption, and the release of cell surface determinants, including alpha2beta1 integrins and CD44, resulted in circumscribed matrix reorganization. Not infrequently, these emerging tube-like paths of least resistance bordered by a dense fiber network facilitated the reorientation and contact guidance of proximate MV3 cells to migrate along the preexisting path. In conclusion, the migration of MV3 cells in 3-dimensional collagen lattices resulted in dynamic tissue reorganization and receptor shedding the consequences of which were directly visualized by combining confocal reflection imaging with immunofluorescence.

Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro.

The invasion and migration occurring in primary neoplastic tissue explants were studied by using a three-dimensional collagen matrix model, subsequent time-lapse videomicroscopy, and computer-assisted cell tracking. We show that not only single cells but groups of clustered cells comprising 5 to more than 100 cells detach from the primary tumor lesion and migrate within the adjacent extracellular matrix. These clusters were highly polarized, resulting in a high directional persistence of migration. Locomoting cell clusters were observed in primary cultures from invasive oral squamous cell carcinomas (6 of 9), ductal breast carcinomas (2 of 3), and rhabdomyosarcoma (1 of 1), whereas normal oral mucosa (0 of 4) was cell cluster negative. Thus, locomoting cell clusters could be a novel and potentially important mechanism of cancer cell invasion and metastasis.

Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines.

The function of dendritic cells (DC) depends on active migration through three-dimensional (3-D) extracellular matrices. We have analyzed the migration of murine DC from different tissue origins within 3-D collagen lattices through the use of time-lapse videomicroscopy and single-cell tracking. Directly after incorporation, 50-90% of DC from the spleen (spDC) and Langerhans cells freshly isolated from the epidermis (fLC) displayed active motility in these matrices. Whereas mature spDC showed multilateral pseudopod dynamics as well as fast and heterogeneous migration, immature fLC displayed a spherical shape with faint membrane processes and very homogenous, slow migration characteristics. In the absence of external stimuli, migration of both, spDC and fLC, vanished after >36 h due to cell death. Maintaining fLC viability by external granulocyte-macrophage colony-stimulating factor or tumor necrosis factor alpha prolonged migration up to 5 days. During this period fLC transformed into mature cells with large dendrites, thereby developing a heterogeneous migration pattern more similar to spDC. In randomly polymerized collagen matrices cell paths were without preferential orientation. In contrast, in artificially aligned lattices directional paths in accordance with the forced fiber orientation were observed. Thus, migration is an inherent property of DC, largely influenced by tissue origin, degree of maturity, and the 3-D structure of the environment.

Gene diagnostics provide new insights into breast cancer prognosis and therapy.

Breast cancer is the most frequent cancer of women in developed countries. It is not surprising, therefore, that major research efforts have been made to improve early detection and treatment strategies directed at this malignancy. In this keynote article, we outline future perspectives on breast cancer genetics, risk factors and prognostic factors, focusing on novel developments relating to the estrogen receptor and the family of ErbB growth factor receptors.

Double-differential PCR for gene dosage estimation of erbB oncogenes in benign and cancer tissues and comparison to cellular DNA content.

Competitive and differential quantitative PCR methods circumvent the limiting factors of PCR which cause poor reproducibility. We describe the development and performance evaluation of another quantitative PCR method, double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. The ratio of band intensities of the PCR products in silver-stained polyacrylamide gels expresses the average gene copy number (AGCN) per cell of the erbB oncogenes. The coefficient of variability (CV) was less than 25% for an AGCN of 1. The PCR data were in correlation to the results from dot blotting. DNA image analysis did not reveal any correlation between DNA content and gene dosage deviation of the erbB oncogenes. The method was applied to normal breast tissue, benign breast diseases, breast cancer tissue and lymph node metastases. We suggest this method as being reproducible, low cost and rapid, and therefore suitable for clinical studies on erbB oncogene dosage estimation.

Prognostic relevance of aberrations in the erbB oncogenes from breast, ovarian, oral and lung cancers: double-differential polymerase chain reaction (ddPCR) for clinical diagnosis.

We have determined the average gene copy numbers (AGCN) of the erbB-1 gene, encoding the epidermal growth factor receptor (EGF-R), the erbB-2 and the erbB-3 genes in breast, ovarian, oral, and lung cancer tissue by using double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. In a retrospective study the AGCN of the erbB genes and the time up to the appearance of metastases were subjected to life-table analysis in 128 women with primary breast cancer. Patients whose breast cancer tissue showed an AGCN for erbB-1 of less than 0.4 and greater then 1.6, as expected from the literature, for erbB-2 of greater than 2.0 and for erbB-3 of less than 1.75 had decreased disease-free survival (DFS). The quotient of erbB-1 and erbB-2 AGCN was the most significant in multivariate Cox analysis followed by nodal status and progesterone receptor status. In extensive studies a similar association between erbB AGCN and metastasis was seen in ovarian cancer and oral cancer, though erbB oncogene aberrations in those entities were not as frequent as in breast cancer. The AGCN of erbB oncogenes may not be of prognostic value in untreated lung cancer patients.

Detection of the metastatic potential of blood-borne and immunomagnetically enriched epithelial cells by quantitative erbB-2 RT-PCR.

A quantitative competitive RT-PCR targeting the specific mRNA of c-erbB-2 is described. It is well known that in many cancers the c-erbB-2 gene dosage is elevated and/or the mRNA is overexpressed leading to a poor prognosis. The recently established method to enrich epithelial cells from peripheral blood made it reasonable and logical to develop a sophisticated competitive RT-PCR method to estimate the c-erbB-2 mRNA load of such cells. By this enrichment method it was discovered that cancer cells can be either isolated as single cells or clusters from patients' blood. The plausibility for their metastatic potential is derived from the measurement of the c-erbB-2 mRNA expression. A SKBR3 cell model suggested that the c-erbB-2 mRNA load is between 168 and 336 molecules per cell. Results of the quantitative competitive RT-PCR method can be used as a rational to determine the efficiency of anti-oncogenic therapies and as a criterion in adjuvant therapy decision.

Lymphocyte locomotion in three-dimensional collagen gels. Comparison of three quantitative methods for analysing cell trajectories.

We evaluated three different quantitative evaluation methods for lymphocyte locomotion in three-dimensional collagen gels: (1) the length of the two-dimensional migration path (distance migrated) was compared to (2) the resulting average displacement from the starting to the end point and (3) the displacement of the furthest migrating population (cells with high displacement). Locomotion of immunomagnetically isolated human CD4+ and CD8+ peripheral blood lymphocytes suspended in type I collagen gels was recorded using time-lapse videomicroscopy. Paths of randomly selected locomoting cells were digitized, reconstructed and quantitatively analysed. For spontaneously locomoting CD4+ and CD8+ lymphocytes (90 min observation period) the mean total distance migrated was 10.0 +/- 3.7 microns/min (CD4+; n = 114 cells) and 5.6 +/- 3.3 microns/min (CD8+; n = 90 cells). The mean displacement from the individual starting point amounted to 1.3 +/- 0.7 micron/min for CD4+ and 1.1 +/- 0.7 micron/min for CD8+ cells, thus representing only 5-25% of the total migration path (index range displacement/distance migrated: 0.13-50%). Incubation with interleukin-8 and/or receptor blocking by monoclonal antibodies against VLA-2 (Gi9) or VLA-4 (HP2/1) integrins significantly altered the mean length of the migration paths for six out of ten different experimental conditions. Average displacement or displacement of the most active cells detected significant changes in two and three out of ten samples. Whereas the interleukin-8 induced locomotory changes were correctly represented by end point determination, relatively slight but significant modulation in lymphocyte behaviour by anti-integrin antibodies was revealed solely by analysis of the complete cell trajectory. In conclusion, the cell trajectory may represent a sensitive method for evaluating induced subtle changes in lymphocyte locomotory characteristics.

An immunological enrichment method for epithelial cells from peripheral blood.

The ability of primary tumours to metastasize accounts for the majority of cancer deaths. The emergence of circulating carcinoma cells in the peripheral blood is supposed to be an indicator for cancer cell spread. We have focused on this phenomenon in order to develop a sensitive technique for enriching epithelial derived cells on the basis of a two-layer density gradient and subsequent immune magnetic cell sorting. Epithelial cells are possess a cytoskeleton containing an assembly of intermediate filaments. During carcinogenesis these filaments do not undergo modifications of antibody binding epitopes such as occur in the protein domains of surface markers. We have developed a two-layer density gradient in which the epithelial cells form a single density band. This was demonstrated by recovery experiments using [3H]thymidine-labelled epithelial cells which showed epithelial cells were enriched within this first step by a factor of 20. In a second step the MACS system was applied. Cells were stained with a performed FITC-conjugated mouse anti-human cytokeratin antibody bound to a rat anti-mouse antibody coupled to superparamagnetic particles (immune paramagnetic separation complex; IPSC) and subjected to high gradient magnetic fields. The two-step procedure was confirmed by dispersing 50 epithelial cells in 5 x 10(5), 5 x 10(6), 5 x 10(7), 5 x 10(8), 5 x 10(9) peripheral blood leucocytes. Specific binding of the preformed IPSC was demonstrated by flow cytometry, confocal laser, fluorescent and electron microscopy. The specificity of the method was further proved by dual staining with IPSC and anti-human PSA antibody of epithelial prostatic cells separated from peripheral blood in vitro. By means of this double-step separation method it was possible to isolate up to 15-20 cells out of 50 epithelial cells originally suspended into 5 x 10(7) to 5 x 10(9) human peripheral blood leucocytes. This represented an enrichment factor between 20,000 and 200,000, depending on the initial cell number. The immunologically captured epithelial cells can be used for further cytogenetic investigations such as in situ hybridization (ISH) and/or polymerase chain reaction (PCR) to detect cancer cell specific gene aberrations. This sensitive combined buoyant density immune magnetic cell separation technique is capable of detecting free carcinoma cells in the peripheral blood.

Differences in the migration capacity of primary human colon carcinoma cells (SW480) and their lymph node metastatic derivatives (SW620).

Two human cell lines, one established from a colon carcinoma (SW480) and the other from its lymph node metastasis (SW620), were compared with respect to their migration capacity employing a three-dimensional collagen matrix and time-lapse video recording. Non-motile cells were characterized by a round shape, whereas motile cells appeared in an elongated form with pseudopodia. The primary tumor cells showed a higher spontaneous locomoting activity than the cells from the metastasis. Using single cell analysis, the distance migrated within 15 h was slightly increased in the presence of hyaluronic acid (HA) in both cell lines. An investigation of the amount of CD44 on the cell surface using the anti-CD44 antibody Hermes-1 showed only minor concentrations of this glycoprotein on cells from the metastasis, whereas a much higher amount was found on cells derived from the primary tumor. The distribution of CD44 on the cell surfaces of HA-treated and untreated cells did not differ as shown by confocal laser scanning microscopy in SW480. The results indicate a restricted influence of HA on migration in the two cell lines.

Prevention of entrance into G2 cell cycle phase by mimosine decreases locomotion of cells from the tumor cell line SW480.

Cellular proliferation of tumor cells is thought to impede migratory activity. Using continuous single cell migration analysis of the colon carcinoma cell line SW480 for up to 72 h, we were able to show that cells locomote constantly and stop only for actual cell division. These findings indicate that proliferation (from G1 phase to early mitosis) and migration do occur simultaneously. The presence of the cell cycle marker Ki-67 in individual migrating cells substantiated this observation. Inhibition of cell cycle progression by mimosine (MIM), a reversible cell cycle blocker, reduced the percentage of migrating cells; release from MIM block restored migratory capacity. The corresponding cell cycle phase distributions were confirmed by flow cytometry. In our test system cell cycle events and migration were shown to occur at the same time. Interference with cell cycle progression reduced migratory activity indicating that migration depends on an unhampered cell cycle.

Influence of hyaluronic acid or phorbol 12-myristate 13-acetate on the migration capacity of a murine lymphoma cell line (Eb) and its metastatic variant (ESb).

The in vitro migration of two murine T cell lymphoma cell lines (Eb and ESb) was studied employing a three-dimensional collagen matrix and time-lapse video recording. In the highly metastatic cell line ESb, which had a low spontaneous locomoting activity, migration could clearly be stimulated by hyaluronic acid (HA) whereas only a small increase was found after incubation with phorbol myristate acetate (PMA). The observed stimulation could be attributed to an increase in recruitment of locomoting cells and not to changes in migration parameters of motile individual cells such as percentage of time locomoting, velocity or distance migrated. Incubation of the low metastatic cell line Eb with HA led to a decrease in migration but blocking of CD44, the principle ligand for HA, by preincubation with an anti-CD44 mAb (KM114), followed by HA exposure increased the locomoting activity significantly. The effect was based on both an increase in recruitment as well as in all migration parameters regarding motile individual Eb cells.

Predictive laboratory diagnostics in oncology utilizing blood-borne cancer cells--current best practice and unmet needs.

The aim of laboratory diagnostics in oncology is to improve the clinical outcome of cancer by allowing earlier detection. Molecular knowledge of cancer should increase the number of risk and prognostic factors and will allow development of methods for detection and elimination of even very small tumors. Thus, the race for the specific tumor antigen in peripheral blood and the race for the blood-borne cancer cell happened simultaneously. The direct detection of the cells which have the highest probability to harbor all the properties mandatory to be life-threatening, conceivably metastatic, would be the most promising way to find the target structure of malignancy. Methods applying enrichment techniques based on density, morphology, tissue specific protein and tumor-associated protein detection enabled multi-parametric analysis of those blood-borne cancer cells. In exemplary studies it was demonstrated that the count of cell clusters positive for the tissue-specific proteins cytokeratin and prostate-specific antigen (PSA) from the peripheral blood of prostate cancer patients and a combination of a tissue-specific protein, a oncogenic receptor protein cytokeratin and p185(c-erbB-2) from the peripheral blood of breast cancer patients is related to the stage of the diseases. Breast cancer patients who presented with cytokeratin/p185(c-erbB-2) positive cell clusters showed a decrease of those cells under adriamycin adjuvant therapy. Nevertheless, additional molecular markers are required to characterize the functional properties of blood-borne cancer cells. Therefore, the genome of the cells can be investigated using a procedure for indirectly detecting aberrations of defined gene locations, i.e. multiplex microsatellite polymerase chain reaction. Up to now, the methods applied to the separation of blood-borne cancer cells are time-consuming and rather expensive. They consist of an initial enrichment step of density gradient centrifugation or buffy coat preparation followed by a specific isolation step using superparamagnetic microbeads coupled to antibodies, filter techniques or multi-parametric flow cytometry. Novel technologies have to be applied using miniaturization, integration and parallel-processing techniques based on those used in the computer industry to overcome the drawbacks.

Migration and metalloproteinases determine the invasive potential of mouse melanoma cells, but not melanin and telomerase.

The biological characteristic cell locomotion and invasion, melanin content, metalloproteinases and telomerase activity were studied in a parental mouse melanoma cell line B16 and two descendents B16BL6 and B16F10. The invasive potential of melanoma cells was assayed in a transwell cell culture chamber. Melanin content was determined by the absorbance value at 470 nm per 10(6) cells. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was adopted to assay the metalloproteinases secretion. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) was used for measuring telomerase activity. The results demonstrated that B16BL6 and B16F10 cells were highly invasive compared to B16 cells, but the melanin content of B16F10 was very low. B16F10 and B16BL6 were hypermotile and secreted much more metalloproteinases than B16. No differences were observed in telomerase activity among the three melanoma cell lines. Invasion of mouse melanoma was closely correlated to tumor cell migration and secretion of metalloproteinases. Melanin content and telomerase activity were phenotypically not related to invasiveness in these three mouse melanoma cell lines.