Low Metastatic Potential Cell Research Articles

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing. Key words: Whole exome sequencing; Hepatocellular carcinoma; Mutations; Metastasis

Mining featured biomarkers associated with vascular invasion in HCC by bioinformatics analysis with TCGA RNA sequencing data

This study aims to identify the feature genes associated with vascular invasion in hepatocellular carcinoma (HCC). Here, the RNA sequencing data related to vascular invasion in The Cancer Genome Atlas (TCGA) database, including 292 HCC patients with complete clinical data were included in our study as the training dataset for construction and E-TABM-36, including 41 HCC patients with complete clinical data was used as the validation dataset. Following data normalization, differentially expressed mRNA and copy number (CN) were selected between with and without vascular invasion samples. A support vector machine (SVM) classifier was constructed and validated in GSE9828 and GSE20017 datasets. Total 59 feature genes were found by the SVM classifier. Using Cox regression analysis, three clinical features, including Patholigic T, Stage and vascular invasion and 6 optimal prognostic genes, including ANO1, EPHX2, GFRA1, OLFM2, SERPINA10 and TKT were significantly correlated with prognosis. A risk score formula was developed to assess the prognostic value of 6 optimal prognostic genes, which were identified to possess the most remarkable correlation with overall survival in HCC patients. By performing in vitro experiments, we observed TKT was significantly increased, but OLFM2 was decreased in high metastatic potential HCC cell lines (SK-HEP-1 and MHCC-97 H) compared with low metastatic potential cell line Huh7 and normal human liver cell line LO2 using western blotting analysis. Knockdown of TKT in MHCC-97H or overexpression of OLFM2 in SK-HEP-1 significantly suppressed cell migration and invasion using transwell assays. Our results demonstrated that TKT and OLFM2 might be novel independent biomarkers for predicting survival based on the presence of vascular invasion in patients with HCC.

Quantitative phase imaging reveals matrix stiffness-dependent growth and migration of cancer cells

Cancer progression involves complex signals within the tumor microenvironment that orchestrate proliferation and invasive processes. The mechanical properties of the extracellular matrix (ECM) within this microenvironment has been demonstrated to influence growth and the migratory phenotype that precedes invasion. Here we present the integration of a label-free quantitative phase imaging technique, spatial light interference microscopy (SLIM)—with protein-conjugated hydrogel substrates—to explore how the stiffness of the ECM influences melanoma cells of varying metastatic potential. Melanoma cells of high metastatic potential demonstrate increased growth and velocity characteristics relative to cells of low metastatic potential. Cell velocity in the highly metastatic population shows a relative stability at higher matrix stiffness suggesting adoption of migratory routines that are independent of mechanics to facilitate invasion. The use of SLIM and engineered substrates provides a new approach to characterize the invasive properties of live cells as a function of microenvironment parameters. This work provides fundamental insight into the relationship between growth, migration and metastatic potential, and provides a new tool for profiling cancer cells for clinical grading and development of patient-specific therapeutic regimens.

Proximity of Metastatic Cells Enhances Their Mechanobiological Invasiveness.

A critical step in metastases formation is cancer-cell invasion through tissue. During invasion, cells change morphology and apply forces to their surroundings. We have previously shown that single, metastatic breast-cancer cells will mechanically indent a synthetic, impenetrable polyacrylamide gel with physiological-stiffness in attempted invasion; benign breast cells do not indent the gels. In solid tumors, e.g., breast cancers, metastases occur predominantly by collective cell-invasion. Thus, here we evaluate the effects of cell proximity on mechanical invasiveness, specifically throughchanges in gel indention. Gel indentation is induced by 56, 33 and 2% (in >1000 cells), respectively, of adjacent high metastatic potential (MP), low MP and benign breast cells, being double the amounts observed in single, well-separated cells. Single cells exhibited a distribution of indentationdepths below 10µm, while adjacent cells also showed a second peak of deeper indentations. The second peak included 65% of indenting high MP cells as compared to 15% in the low MP cells, illustrating the difference in theirinvasiveness. Thus, proximity of the metastatic cells enhances their mechanical ability to invade, demonstrating why collective cancer-cell migration is likely more efficient. This could potentially provide a rapid, quantitative approach to identify metastatic cells, and to determine their metastatic potential.

Origin of active transport in breast-cancer cells

Living cells are active systems, continuously adapting to respond to their environment. The dynamic cytoskeleton and especially the molecular motors acting on it provide the cell with its remodeling capabilities and allow active transport within the cell. While active transport in living cells has been well-documented, the underlying mechanisms have not been determined. Hence, we systematically target the cytoskeleton, molecular motors, and ATP energy related processes to determine their roles in particle transport. We perform intracellular particle tracking in low and high metastatic potential (MP) breast-cancer cells and analyze particle motion through the mean square displacement and other powers of the displacement. From those experiments, we show that the motion in both cell types is actively driven by fluctuating microtubules and their associated molecular motors. In the two cells, however, the relative importance of the mechanisms varies. In the low MP cells, particles are mostly transported by motors and likely remain on a single microtubule. In those cells, microtubule fluctuations cause intermittent jumps and actin typically hinders motion in the dense intracellular microenvironment. In contrast, particle motion in the high MP cells is driven by indirect motor-transport or by jumping between microtubules and is highly impacted by fluctuations of the microtubules. Thus, we are able to provide insight into mechanisms driving active transport in living cells, using intracellular particle tracking.

Overexpression and cytoplasmic accumulation of Hepl is associated with clinicopathological parameters and poor prognosis in non-small cell lung cancer

Hepl, first described in 2008, is the fourth member of the Crk-associated substrate (CAS) family and is specifically expressed in the lung. Compared to other CAS proteins, Hepl has a varying effect on cell migration in different cell types. We speculated that Hepl may play a role in lung cancer invasion and metastasis. We quantified the expression and subcellular localization of Hepl in 143 non-small cell lung cancer (NSCLC) tissues, adjacent noncancerous tissues, and eight lung cancer cell lines using Western blotting, immunohistochemistry, and immunofluorescent staining. Expression of Hepl was correlated with the clinicopathological features of NSCLC. Hepl was overexpressed in 72.3% (103/143) of the NSCLC tissues, compared to the adjacent noncancerous lung tissues (P = 0.022). Overexpression of Hepl was associated with lymph node metastasis and high TNM stage (P = 0.005 and P = 0.045, respectively). Kaplan-Meier survival curves and the log-rank test indicated that overexpression of Hepl correlated with poorer overall survival in NSCLC (P < 0.001), and Cox regression analysis demonstrated that overexpression of Hepl was an independent prognostic factor in NSCLC. Furthermore, cytoplasmic accumulation of Hepl was observed in a high metastatic potential lung cancer cell lines (H1299 and BE1), but not in low metastatic potential cell lines (LTE and A549). This study reveals that Hepl is overexpressed in the nucleus and aberrantly accumulates in the cytoplasm of NSCLC cells, and indicates that Hepl may play a role in the progression of lung cancer, including lymph node metastasis and TNM stage. Additionally, Hepl may be a useful prognostic factor in lung cancer.

Expression of metadherin/AEG-1 gene is positively related to orientation chemotaxis and adhesion of human hepatocellular carcinoma cell lines of different metastatic potentials

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). However, the mechanism through which a primary HCC cell develops into a metastatic phenotype is not well understood. The purpose of this study was to examine the correlation between metadherin (MTDH)/astrocyte elevated gene-1 (AEG-1) expression in HCC cell lines of different metastatic potentials and such metastatic phenotypes as orientation chemotaxis and adhesion. MTDH/AEG-1 expression was detected by RT-PCR and western blotting in HCC cell lines (HepG2, Huh7, Sk-HEP-1, MHCC-97H). Distribution of MTDH/AEG-1 was observed by immunofluorescence staining and confocal laser scanning microscopy. The abilities of orientation chemotaxis and adhesion and the index of interaction between HCC cell lines and microvascular endothelial cell lines (MVECs, including HUVECs and HPMECs) were measured by chemotaxis assay and adhesion assay, respectively. The results showed that MTDH/AEG-1 protein expression was significantly higher in high metastatic potential cancer cell lines (Sk-HEP-1, MHCC-97H) than in low metastatic potential cell lines (HepG2, Huh7) (P<0.05). The MTDH/AEG-1 protein was localized in the perinuclear region of HCC cells. Furthermore, the abilities of orientation chemotaxis and adhesion of HCC cells to HPMECs were increased as compared with those of HCC cells to HUVECs (P<0.05). The abilities of orientation chemotaxis and adhesion were much stronger in Sk-HEP-1 and MHCC-97H cells with MTDH/AEG-1 highly expressed than in HepG2 and Huh7 cells with MTDH/AEG-1 lowly expressed (P<0.05). These results suggested that the expression of MTDH/AEG-1 gene in HCC cell lines of different metastatic potentials was closely positively related to the abilities of orientation chemotaxis and adhesion of HCC cells. It was deduced that MTDH/AEG-1 might play a pivotal role in the lung-specific metastasis of HCC, which may be mediated through orientation chemotaxis and adhesion abilities of HCC cells. MTDH/AEG-1 may serve as a potential therapeutic target for HCC.

Expression of osteopontin in different metastatic potential uveal melanoma and its significance

Background Osteopontin is highly expressed in many metastatic tumors,but its expression in uveal melanoma and the relationship of osteopontin with metastasis is still under investigation. Objective This study was to investigate the relationship between osteopontin expression and uveal melanoma metastatic potential.Methods Fifty samples of uveal melanoma were collected in Beijing Tongren Hospital.The expressions of osteopontin in the samples were detected using immunohistochemistry,and expressions of osteopontin mRNA in different metastatic potential uveal melanoma cell lines (MU M-2B,C918,OCM-1A) were analyzed using quantitative polymerase chain reaction (Q-PCR). Results Osteopontin was positively expressed in 10 samples of 13 patients with metastasis and 14 samples of 37 patients without metastasis,showing a significant difference between them (x2=5.888,P=0.000).There were 11 samples and 13 samples exhibiting the positive expression of osteopontin in 15 epithelial type and 35 non-epithelial type of uveal melanoma respectively with the significant difference (x2=5.510,P =0.000).The positive expressing numbers of osteopontin were 20 tumors of 30 affected tumors and 4 tumors of 20unaffected ciliary tumors with the significant difference (x2 =10.470,P =0.000 ).No significant differences in the expression of osteopontige were found between different gender,age,eyes,largest basal diameters and invasion of scleral canal( P =0.536,0.256,0.802,0.848,0.555 ).The relative expression value of osteopontin mRNA was in turn 1.00±0.04,0.91 ±0.03,0.08±0.01 in MUM-2B,C918,OCM-1A with the significant difference among three cell lines( F=33.135,P＜0.05).The osteopontige mRNA expression was significant enhanced in high metastatic potentialcell lines( MUM-2B,C918 ) compared with low metastatic potential cell lines( OCM-1 A) (P＜0.05 ),but no significant difference was seen between two high metastatic potential cell lines(MUM-2B,C918) (P=0.804). Conclusions Osteopontige expression is associated with the uveal melanoma cell line invasion and metastasis potential.These results imply that osteopontige expression maybe an indicator of uveal melanoma invasion and metastasis ability. Key words: Uveal melanoma; Osteopontin; Invasion and metastasis

Profiling analysis of differential gene expression between breast cancer cell sublines with high- and low-metastatic potential

Objective To establish variant cell sublines expressing green fluorescent protein with high- and low-metastatic potential, and investigate gene expression profile in these cell sublines in order to find new candidate genes related to metastasis of breast cancer to bone. Methods Twenty μg total RNA of MCF7 cell sublines A2 and C6 was extracted, purified to mRNA and then reversely transcripted to cDNA probe which labeled with Cy5 and Cy3 fluorescence respectively. The two samples were hybridized with the cDNA microarray chips containing 7458 human target genes. The hybridization signals were scanned by Generation Ⅲ array scanner and obtained data were analyzed using Imagequant 5. 0 and Array Vision 6. 0 software. Results There were significant differences in gene expression between A2 and C6 cell sublines.Among the target genes, 767 differentially expressed genes were identified. As compared to A2, 480 of 767 differentially expressed genes were up-regulated and the remaining 287 were down-regulated. Conclusion There were different gene expression profiles between high- and low-metastatic potential cell sublines.These genes may provide important clues for finding genes related to metastasis of breast cancer to bone. Key words: Breast carcinoma; Metastasis; cDN A microarray; Gene

Expression of Transgelin in hepatocellular carcinoma is correlated with its prognosis

Objective To investigate the correlation of the level of Transgelin expression in hep-tacellular carcinoma with its recurrence and prognosis. Methods The expression of Transgelin in dif-ferent metastatic potential hepatocellular carcinoma cell lines and cancerous lesions, tissue adjacent to cancer lesions and normal liver tissues collected during the operation were detected by Western-blot-ting. The level of Transgelin expression in these clinical samples was accessed by immunohistochemi-cal staining. The correlation between the level of Transgelin expression and the prognosis was analyzed by means of log-rank test and Kaplan-Meier analysis. Results Western blotting test showed that the expression level of Transgelin was significantly higher in high metastatic potential HCC cell line than in low metastatic potential cell line. Meanwhile, it was higher in tumor tissue than in tissue adjacent to cancer lesions or normal liver tissue. Irnmunohistochemical staining test showed the similar result. Pa-tients with positive expression of Transgelin had worse survival and tumor free survival than those with negative ones (P<0.01). Conclusion The expression of Transgelin in hepatocellular carcinoma is correlated with its metastatic potential and this protein could be the biomarker of the prognosis. Key words: Carcinoma hepatocellular; Transgelin; Prognosis

A decade's studies on metastasis of hepatocellular carcinoma.

Metastasis remains one of the major challenges before hepatocellular carcinoma (HCC) is finally conquered. This paper summarized a decade's studies on HCC metastasis at the Liver Cancer Institute of Fudan University. We have established a stepwise metastatic human HCC model system, which included a metastatic HCC model in nude mice (LCI-D20), a HCC cell line with high metastatic potential (MHCC97), a relatively low metastatic potential cell clone (MHCC97L) and several stepwise high metastatic potential cell clones (MHCC97H, HCCLM3, and HCCLM6) from their parent MHCC97 cell. Endeavors have been made for searching human HCC metastasis-related chromosomes/proteins/genes. Monogene-based studies revealed that HCC invasion/metastasis was similar to that of other solid tumors, and the biological characteristics of small HCC were only slightly better than that of large HCC. Using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), genotyping, cDNA microarray, and 2-dimensional gel electrophoresis, we obtained some interesting results. In particular, in collaboration with the National Institute of Health (NIH) in the United States, we generated a molecular signature that can classify metastatic HCC patients, identified osteopontin as a lead gene in the signature, and found that genes favoring metastasis progression were initiated in the primary tumors. We also found that chromosome 8p deletion, particularly in the region of 8p23, was associated with HCC metastasis. Cytokeratin 19 was identified as one of the proteins, which was found in MHCC97H, but not in MHCC97L cells. Experimental interventions using the high metastatic nude mice model have provided clues for the prevention of HCC metastasis. Translation from workbench to bedside demonstrated that serum VEGF, microvessel density, and p53 scoring may be of value for the prediction of postoperative metastatic recurrence. Interferon alpha proved effective for the prevention of recurrence both experimentally and clinically. In conclusion, HCC metastasis that probably initiated in the primary tumor is a multigene-involved, multistep, and changing process. The further elucidation of the mechanism underlying HCC metastasis will provide a more solid basis for the prediction and prevention of the metastatic recurrence of HCC.

The level of cell surface beta1,4-galactosyltransferase I influences the invasive potential of murine melanoma cells.

Beta1,4-Galactosyltransferase I (GalT I) is localized on the leading lamellipodia of migrating cells, where it associates with the cytoskeleton and facilitates cell spreading and migration on basal lamina matrices. It has previously been reported that a variety of highly metastatic murine and human cell lines are characterized by elevated levels of cell surface GalT I, although the intracellular biosynthetic pool is similar between cells of high and low metastatic potential. In this study, we examined whether the elevated expression of surface GalT I characteristic of metastatic cells is instructive or incidental to their metastatic behavior by altering the expression of surface GalT I and by the use of GalT I-specific perturbants. Surface GalT I levels were positively and negatively altered on murine melanoma cells by either overexpressing full-length GalT I or by homologous recombination, respectively. The consequences of altered surface GalT I expression on cell invasion in vitro and lung colonization in vivo were determined. Increasing surface GalT I expression on cells of low metastatic potential to levels characteristic of highly metastatic cells recapitulated the highly invasive phenotype in vitro. Alternatively, decreasing surface GalT I expression on highly metastatic cells to levels characteristic of low metastatic cells reduced their invasive behavior in vitro and metastatic activity in vivo. Within the physiological range of surface GalT I expression, the invasive potential of each clonal cell line correlated strongly with the level of surface GalT I expressed. As an independent means to assess the involvement of surface GalT I in metastatic behavior, cells were pretreated with two different classes of surface GalT I perturbants, a competitive oligosaccharide substrate and a substrate modifier protein. Both perturbants inhibited metastatic colonization of the lung, whereas control reagents did not. Finally, as reported by others, surface GalT I on metastatic cells selectively interacted with one glycoprotein substrate, or ligand, of approximately 100 kDa, the identity of which remains obscure. These results show that the elevated expression of surface GalT I characteristic of highly metastatic cells contributes to their invasive phenotype in vitro and to their metastatic phenotype in vivo.

Metastasis of human colon cancer is altered by modifying expression of the beta-galactoside-binding protein galectin 3.

Galectin 3 is a beta-galactoside-binding protein whose expression has been correlated with advanced tumor stage in the colon, but direct evidence for a role in metastasis is lacking. The current study was designed to more directly establish the role of galectin 3 in colon cancer metastasis. Galectin 3 levels were manipulated in human colon cancer cells using eukaryotic expression constructs designed to express the complete galectin 3 complementary DNA in either the sense or antisense orientation. Liver colonization was assessed in athymic mice after splenic-portal inoculation or after spontaneous metastasis during cecal growth. Introduction of galectin 3 antisense into metastatic colon cancer cells (LSLiM6, HM7) resulted in a significant reduction in galectin 3-specific messenger RNA and total and cell surface galectin 3 protein. Conversely, stable integration of galectin 3 in the sense orientation resulted in an increase in cellular and cell surface galectin 3 in cells of low metastatic potential (LS174T). Reduction in galectin 3 levels was associated with a marked decrease in liver colonization and spontaneous metastasis by LSLiM6 and HM7 cells, whereas up-regulation of galectin 3 resulted in increased metastasis by LS174T cells. This study provides direct evidence that galectin 3 plays an important role in colon cancer metastasis.

The role of urokinase and urokinase inhibitor in tumour cell metastasis

In this paper we summarize experiments in which murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for prep rourokinase. B16-F1 cells selected for their secretion of high amounts of the human urokinase gene product (3- to 4-fold higher plasminogen activator activity than control cells) formed between 4- and 16-fold greater numbers of lung tumours in C57BL mice after tail vein injection. In correlative results, highly malignant B16-F10 melanoma cells transfected with a urokinase antisense sequence showed a significant inhibition of endogenous urokinase expression and a corresponding significant decrease in the number of tumours formed after tail vein injection. The human urokinase gene product expressed by the transfected cells does not bind to murine cell surface urokinase receptors, and the results may provide direct evidence for a role of secreted (non-surface bound) urokinase in the extravasation steps of tumour cell metastasis. These human urokinase secreting cells also showed a greater ability to spontaneously metastasize from a primary footpad tumour to the mouse lung. Extending the study of urokinase to the role of its natural inhibitor, plasminogen activator inhibitor 2 (PAI-2), we determined the constitutive and phorbol ester inducibility of the mRNA for PAI-2 and urokinase in human A375 melanoma cell lines and human prostate PC3 carcinoma cell lines of different metastatic potentials. Analysis of the changes in PAI-2 and urokinase mRNA levels indicate that variation in the levels of PAI-2 may be more important than urokinase in explaining the different metastatic abilities of A375 and PC3 sublines. Urokinase activity may promote metastasis by its initiation of a specific proteolysis pathway leading to the activation of metalloproteinases. We have previously shown that the metalloproteinase inhibitor, recombinant tissue inhibitor of metalloproteinases (rTIMP), inhibits B16 melanoma cell lung colonization.

Involvement of urokinase and its receptor in the invasiveness of human prostatic carcinoma cell lines.

We have investigated the role of urokinase (UK) and its cell-surface receptor in determining the invasiveness of prostate cancer cells. Three human cell lines, DU-145, PC-3 and LNCaP, that differ in androgen-responsiveness and growth characteristics, were tested. Analysis of the conditioned medium by an enzyme-linked immunosorbent assay showed secretion of UK by DU-145 (63 ng/mL/10(6) cells/48 hr) and PC-3 (682 ng/mL/10(6) cells/48 hr), but absence of secretion by LNCaP cells. Western blot analysis and enzyme activity assay of the conditioned medium confirmed these results. Scatchard analysis of radioligand binding with acid pretreated cells showed the presence of a single population of high affinity UK receptors on DU-145 cells (93,000 sites/cell, Kd = 0.9 nM) and PC-3 cells (25,000 sites/cell, Kd = 1.0 nM) but not on LNCaP cells. DU-145 and PC-3 cells were found to be highly invasive in in vitro invasion assays: 4.5 +/- 0.5% and 6.5 +/- 0.5%, respectively, of total tumor cells (approximately 2 x 10(5)) had penetrated reconstituted basement membrane (Matrigel) in a 72 hr incubation in serum-free growth medium. Under similar conditions, less than 0.25% LNCaP cells invaded Matrigel. The data indicate that androgen unresponsive, aggressive prostate tumor cells of high metastatic potential, DU-145 and PC-3, secrete UK and display cell-surface UK receptors, fully charged with the protease. Conversely, relatively indolent LNCaP cells of low metastatic potential do not secrete UK nor do they possess its binding sites. UK receptor antagonists, UK 12-32 and UK 6-135, which compete with labeled UK for binding to prostatic cells but do not inhibit cellular proliferation or UK secretion, markedly reduced DU-145 and PC-3 cell invasion (80-85% inhibition), thereby suggesting an important role of receptor-bound UK in prostate tumor cell invasion.

The regulation of cyclic AMP production and the role of cyclic AMP in B16 melanoma cells of differing metastatic potential

The nature of the relationship between agonist-stimulated cyclic AMP production and metastatic potential was examined in detail for four B16 melanoma cell lines of varying metastatic potential. Highly metastatic cells (B16 F10C1) appeared to differ from cells of low metastatic potential (B16 F1C29) in the degree to which cyclic AMP production in intact cells was stimulated by protein kinase C activation. No significant difference was found in the adenylate-cyclase enzyme activities of the broken cells, irrespective of the agonist used, or in the distribution of cyclic AMP between the intracellular and extracellular compartment. Although B16F1, F10 and F10C1 cells all produced equally pigmented tumors in vivo, the cells differed in their melanogenic response to cyclic AMP elevating agents in vitro: the least metastatic cells produced least agonist-induced cyclic AMP but this induced greatest tyrosinase activation and melanin production in vitro; conversely, the more metastatic cells produced more cyclic AMP but less tyrosinase activation and melanin production in response to agonist stimulation. Thus, agonist-stimulated cyclic AMP production does not appear to be coupled to the differentiated function of melanogenesis for highly metastatic B16 melanoma cells.

Tumor metastasis-associated heparanase (heparan sulfate endoglycosidase) activity in human melanoma cells

We discovered previously a tumor metastasis-associated heparan sulfate (HS)-degrading endoglycosidase in mouse melanoma cells that is a unique endo-β-glucuronidase (heparanase) capable of specifically cleaving HS at intrachain sites (Nakajima et al., J. Biol. Chem., 259 (1984) 2283. Using unmodified- and chemically modified-HS and heparin substrates we demonstrate that human Hs939 and mouse B16 melanoma cells possess a HS-degrading endoglycosidase of similar specificity. Melanoma heparanase showed high activity against N-desulfated N-acetylated HS, and we therefore synthesized a solid-phase heparanase substrate crosslinking partially N-desulfated N-[ 14C] acetylated HS to agarose gel beads via one covalent linkage. Using this solid-phase substrate 15 human malignant melanoma cell lines were assayed for heparanase activity. All of the melanoma cells tested had heparanase activity, and almost all possessed activities comparable or greater than that of the murine B16-F1 melanoma line. Human A375 melanoma variants of high lung metastatic potential in athymic nude mice had significantly higher heparanase activities than did A375 parental cells of low metastatic potential.

Interactions of tumor cells with intact capillaries: a model for intravasation.

The interactions of metastatic variants of the B16 and K-1735 melanoma cell lines with intact capillaries isolated from rabbit brain were studied in vitro. The abilities of various cells to attach to and flatten out on the surface of blood vessels were monitored with phase contrast and scanning electron microscopy. In general, cells highly metastatic in vivo were capable of attaching to and flattening out on the surface of capillaries at a faster rate than cells of low metastatic potential or normal cells. In addition, K-1735 melanoma cells and normal fibroblasts were labeled with 125I-iododeoxyuridine, incubated with capillaries, and subsequently passed through filters. This procedure separated unattached cells from cells attached to capillaries. This assay provided quantitative information on the adhesion of metastatic tumor cells and normal cells to capillaries. Tumor cells attached to capillaries in significantly greater numbers than normal cells. Cells with high metastatic potential attached to the capillaries in greater numbers than cells with low metastatic potential, but not significantly. This model may be useful for elucidating some of the mechanisms involved in tumor cell attachment and penetration of the capillary wall during intravasation in vivo.