CD44 and its role in tumour progression and metastasis.
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Animals↗
Biomedical subjects
I R Hart
Publications and source records attributed to I R Hart.
Expression and function of the CD44 glycoprotein in melanoma cell lines.
We have analysed a panel of murine and human melanoma cell lines for expression of the glycoprotein CD44. All 12 cell lines examined expressed CD44 at their cell surfaces, as demonstrated by fluorocytometric analysis using monoclonal antibodies (MAbs) IM7 and F10.44.2. Northern analysis revealed three transcript sizes that were 4.5, 2.2, and 1.5 kb in the human cell lines and 4.5, 3.0, and 1.5 kb in the murine cell lines. Levels of mRNA did not correlate with level of surface expression, which was highly variable between the cell lines. RT-PCR analysis of mRNA revealed that the major band identified was the expected 792 bp fragment indicative of the CD44H haemopoietic form, compared to a 1194 bp form found in the human colorectal adenocarcinoma cell line HT29, indicative of the CD44E epithelial form. There was no evidence of variant CD44 mRNA in our panel of melanomas. Functional assays revealed no clear correlation between the level of cell surface CD44 and the ability of the melanoma cell lines to adhere to hyaluronate. Rather adherence appeared to relate to the activation status of CD44 on the different cell lines as a consequence of MAb stimulation (e.g. the 1735P line demonstrated a 46.2 +/- 5.7% adherence in an inactivated state versus 62.4 +/- 5.6% adherence in an activated state to 5 mg/ml hyaluronate) and suggests that the functional capacity of CD44 expressed by melanoma cells may be modified more by activation state than by RNA splicing.
Adenocarcinoma↗
Differential induction of 12-O-tetradecanoylphorbol-13-acetate sequence gene expression in murine melanocytes and melanoma cells.
We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281-3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of TIS gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.
3T3 Cells↗
Cell and tissue responses of a murine tumour to phthalocyanine-mediated photodynamic therapy.
Mice bearing a subcutaneously growing tumour (Colo 26) were injected intravenously with the photosensitiser chloroaluminum sulphonated phthalocyanine (5 mg/kg) 24 h prior to irradiating the tumour with laser light (675 nm; 50mW, 100 J/tumour). Energy status of the tumour, as assessed by the loss of high energy phosphates in the 31P-nuclear magnetic resonance spectra, was altered dramatically following treatment, such that the ATP fell to undetectable levels within 1 h of light irradiation. However, assessment of the clonogenic capacity of neoplastic cells isolated from dissociated tumours showed that these rapid changes in cellular metabolism were not reflected in similar rapid changes in cell viability. Reductions in clonogenic capacity, which fell to less than 0.1% of control values at 24h postirradiation, closely mirrored those resulting from the cessation of vascular perfusion. Evaluation of tumour blood flow, using the technique of hydrogen washout, showed that the treatment protocol evoked a gradual and selective reduction in flow within the tumour resulting in complete vascular stasis by approximately 5 h after treatment. The results indicate that while chloroaluminum sulphonated phthalocyanine-mediated photodynamic therapy caused early metabolic damage in neoplastic cells, loss of viability paralleled the induction of complete inhibition of vascular flow in the tumour.
Animals↗
Inhibitors of nitric oxide synthase selectively reduce flow in tumor-associated neovasculature.
1. The effects of L-arginine analogues, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA) and methylene blue on blood flow in a murine adenocarcinoma and melanoma have been investigated. 2. Sponge implants in Balb/c and C57/BL mice were used to host proliferating tumour cells while the washout of 133Xe was employed to assess local blood flow in the implanted sponges. 3. Pharmacological inhibition of nitric oxide (NO) reduced blood flow in both tumours but this effect was reversed by administration of L-arginine. 4. In marked contrast, the effect of these same NO inhibitors on the blood flow in sponge-induced non-neoplastic granulation tissue was negligible. 5. These results strongly suggest that: (a) flow in tumour vessels is modulated by nitric oxide which maintains a dilator tone in neoplastic tissue; (b) the constrictor activity (as monitored by an increase in t1/2 of 133Xe) of NO inhibitors may be attributed to the removal of such dilator tone; (c) many of the abnormalities described in tumour vasculature, such as hyporeactivity or unresponsiveness to vasoactive mediators and maximum vasodilation, may be due to an increase in NO synthesis in cancers.
Adenocarcinoma↗
Isolation and characterization of human melanoma cell variants expressing high and low levels of CD44.
Variants of the human melanoma cell line LT5.1 were selected for high and low expression of the Mr 90,000 CD44 glycoprotein by using the Hermes-3 monoclonal antibody combined with fluorescence-activated cell sorting. Cells were single cell cloned and clones of CD44 high-expressing and CD44 low-expressing phenotype were isolated. The variants, which exhibited up to a 7-fold difference between high and low expression, have maintained a stable phenotype over a period of 3 months in tissue culture. Northern blot analysis of mRNA from the different clones showed correlation of levels of transcripts with fluorescence-activated cell sorting analysis data. Wound migration assays, utilizing the different clones, showed that the low-expressing clones manifested less motility than did cells showing high levels of CD44. Homotypic aggregation of cells was increased in those cells expressing high levels of CD44, and these variants were also better able to adhere to hyaluronate substrates. All of these activities were inhibited by the presence of anti-CD44 antibody. When injected i.v. into nu/nu BALB/c mice, the low-expressing clones gave significantly fewer lung nodules than the high-expressing clones, although the two variant types did not differ in their capacity to form s.c. tumors in similar mice. These results suggest that the CD44 molecule, possibly as a function of its activities as a hyaluronate receptor, may play a vital role in determining the fate of hematogenously disseminating melanoma cells.
Animals↗
Integrin expression in human melanoma cell lines: heterogeneity of vitronectin receptor composition and function.
Ten human melanoma cell lines were examined for integrin-receptor expression using a panel of antibodies directed against different integrin subunits. Considerable heterogeneity was detected for levels of expression of 7 integrins, including the alpha v beta 3 vitronectin receptor where a correlation between tumorigenic capacity in athymic nude mice and alpha v beta 3 levels was found. Detailed analysis of the vitronectin receptor on these lines revealed heterogeneity of composition. In one cell line, VUP, an alpha v beta 1 association was detected and, by antibody-inhibition studies, this receptor was shown to bind vitronectin as its ligand. In another line, DX3, evidence was obtained which indicated that apart from the alpha v beta 3 receptor the alpha v was able to associate with another beta subunit which was not beta 3. The existence of these alternative forms of the vitronectin receptor in this small sample of tumours of common origin might explain why the capacity to bind to fibrinogen and vitronectin substrates by these cells did not necessarily correlate with alpha v beta 3 levels.
Animals↗
Influence of tumor-derived interleukin 1 on melanoma-endothelial cell interactions in vitro.
Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.
Antigens, CD↗
Protein kinase C down-regulation, and not transient activation, correlates with melanocyte growth.
The nontumorigenic, immortal line of murine melanocytes, Mel-ab, requires the continual presence of biologically active phorbol esters for growth (R.E. Wilson et al., Cancer Res., 49:711-716, 1989). Comparable treatments of B16 murine melanoma cells result in partial inhibition of cell proliferation. The role of protein kinase C (PKC) in the modulation of growth of cells from these two melanocytic cell lines has been investigated. Significant levels of PKC were present in quiescent Mel-ab cells as determined by Western blotting, whereas no immunoreactive protein was detected in cell extracts from either proliferating Mel-ab or B16.F1 cells. Phosphorylation of a Mr 80,000 protein, which by one- and two-dimensional gel analysis comigrated with the known Mr 80,000 protein substrate of PKC in fibroblasts, was induced in 12-O-tetradecanoylphorbol-13-acetate-stimulated quiescent Mel-ab cells but not in proliferating Mel-ab cells or B16.F1 melanoma cells. Direct measurement of PKC activity in these cells demonstrated a 10-fold greater level of activity in quiescent Mel-ab cells (262 +/- 50 pmol/min/mg SD) compared with growing cells (22.8 +/- 11.8 pmol/min/mg SD). An intermediate level of activity was detected in proliferating B16.F1 melanoma cells (148.5 +/- 20.4 pmol/min/mg SD). The subcellular distribution of PKC was dependent upon the growth state of the cells such that quiescent Mel-ab cells displayed a higher level of activity in the cytosol, whereas growing Mel-ab cells displayed greater activity in the particulate fraction. Like many other transformed lines, B16.F1 melanoma cells constitutively expressed the majority of enzyme activity in the particulate fraction. Measurement of [3H]phorbol ester binding in intact cells paralleled the PKC activation data such that quiescent Mel-ab cells displayed binding of 1612 +/- 147 cpm/10(6) cells, whereas proliferating Mel-ab and B16.F1 melanoma cells displayed binding of 652 +/- 28 and 947 +/- 81 cpm/10(6) cells, respectively. Membrane-permeant diacylglycerol analogues, which activated but did not down-regulate PKC, were devoid of growth-stimulating effects on melanocytes, even in the presence of the specific diacylglycerol kinase inhibitor, R59022. Together, these data show that PKC down-regulation, and not activation, correlates with the growth of melanocytes in culture.
Animals↗
Cell adhesion receptor expression during melanoma progression and metastasis.
Many steps in melanoma metastasis involve cell-cell or cell-matrix adhesive interactions. The surface molecules which mediate these processes therefore play an important role in regulating melanoma dissemination and their level of expression may alter during the course of tumor progression. Human melanocyte strains and melanoma cell lines have been characterised with regard to levels of cell surface receptors of the integrin family. Increased amounts of at least two integrins, VLA-4 (alpha 4 beta 1) and VnR (alpha v beta 3), appeared to correlate with progression in this tumor, type. A novel VnR composed of an alpha v beta 1 association has been observed in one melanoma cell line and there is the possibility that heterogeneity of integrin composition could affect biological behavior of these tumors. CD44, a cell surface glycoprotein which functions as the major receptor for hyaluronate, is another molecule whose expression increases in transformed cells of the melanocytic lineage. Iterative sorting on the FACS for stable variants, of both human and murine melanomas, expressing low and high levels of CD44 established that lack of expression of this molecule correlated with impaired ability to form pulmonary tumor nodules subsequent to i.v. injection into appropriate recipient mice. These findings illustrate that an understanding of the regulation of melanoma adhesion receptors could provide insights into the process of tumor spread.
Humans↗
Tumour angiogenesis and metastasis.
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Breast Neoplasms↗
Potential use of a charge-coupled device camera for imaging fluorescent dyes in vivo.
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Animals↗
Divergent effects of flavone acetic acid on established versus developing tumour blood flow.
Flavone Acetic Acid (FAA) exerts much of its effect by reducing tumour blood flow. Previous studies on FAA-induced changes in blood flow have used established tumours with a functional microvasculature. Using radioactive Xenon(133Xe) clearance to monitor local blood flow we show that the effects of FAA are dependent on the presence of this functional microvasculature with no evidence that FAA inhibits the actual development of tumour microcirculation. Thus, administration of multiple doses of FAA around the time of tumour cell injection failed to diminish t1/2 values of 133Xe (e.g. t1/2 16 min for FAA vs 14 min for saline controls at 10 days) or to affect tumour volumes (5.55 +/- 0.06 cm3 in FAA-treated animals vs 5.7 +/- 1.3 cm3 in controls at 25 days). In marked contrast a single dose of FAA (200 mg kg-1 body weight) 2 weeks after tumour cell injection dramatically extended t1/2 times (47 min for FAA vs 7 min for controls; P less than 0.001) and significantly reduced tumour burden. This effect is specific for tumour microvasculature and is not directed simply at new vessels since a similar treatment of animals with implanted-sponge-induced granulation tissue had no effect on t1/2 times (6.8 +/- 1.1 min for FAA at 200 mg kg-1 vs 7.2 +/- 1.0 min for saline-treated controls.
Animals↗
Identification of genes controlling metastatic behaviour.
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Animals↗
Photocytotoxic efficacy of sulphonated species of aluminium phthalocyanine against cell monolayers, multicellular spheroids and in vivo tumours.
The problem of relying solely on in vitro data to predict photosensitiser efficacy was demonstrated by examining the uptake and the ability to mediate photocytotoxicity of mono-, di-, tri- and tetra-sulphonated species of chloroaluminium phthalocyanine (AlS1-4Pc) in monolayer cultures of murine Colo 26 cells and in both monolayer and spheroid cultures of human WiDr cells. Cells treated in vitro, whether in monolayer or as spheroids, with the less sulphonated derivatives, AlS1Pc and AlS2Pc, were more susceptible to photocytotoxicity than those treated with AlS3Pc, cells treated with AlS4Pc were even less susceptibile to the cytotoxic effects of light irradiation. Generally these results mirrored the cellular uptake in vitro. When WiDr spheroids were increased in size from 250 microns to 500 microns there was a reduction in uptake of AlS1Pc and AlS2Pc which was reflected by the decreased sensitivity of the larger spheroids to the effects of light irradiation. AlS1Pc had no effect against Colo 26 cells growing as s.c. tumours in syngeneic BALB/c mice; whereas AlS3Pc, AlS2Pc and AlS4Pc produced significant reductions in tumour weights 5 days post laser light irradiation. Of these, AlS2Pc had the most dramatic effect on the colony forming efficiency of tumour cells recovered 24 h after PDT. While, despite their effects on tumour size, AlS3Pc and AlS4Pc scarcely affected the subsequent viability of cells from dissociated tumours. Thus the in vitro efficacy of the sulphonated species of phthalocyanines is not necessarily predictive of their in vivo effectiveness.
Animals↗
Tumor cell progression and differentiation in metastasis.
The development and evolution of tumors is regulated by both genetic and epigenetic events. It is thought that these processes tend to drive neoplastic development in opposing directions so that tumor progression, predominantly as a consequence of mutational events, leads to increasing tumor aggression. Conversely the induction of differentiation, largely through epigenetic mechanisms, tends to cause tumors to evolve to a more benign phenotype. However, these generalizations are a simplistic view of a complex dynamic event where both processes can be overlaid within a single neoplasm. Using malignant melanoma as a model system the alterations in gene expression and their effects upon metastatic dissemination, that accompany some of these changes, both natural and induced, are described.
Animals↗
Role of tumor necrosis factor in flavone acetic acid-induced tumor vasculature shutdown.
Flavone acetic acid (FAA), a novel investigational antitumor agent, has been shown to cause early vascular shutdown in several experimental murine tumors, and this phenomenon is believed to be crucial to FAA's antitumor effects. However, the basis of this FAA-induced tumor vascular shutdown is unknown. In this study a radioactive tracer-clearance technique has been used as an objective indication of tumor blood flow to show that i.p. administered FAA induces a progressive and sustained reduction in blood flow in a colon 26 tumor growing s.c. in syngeneic mice. As early as 1 h after administration, there was a significant increase in the t1/2 clearance value for intratumorally injected 133Xe, reaching a peak at 3 h (117.3 +/- 36.4 versus 7.8 +/- 0.85 min for controls). Significant inhibition of blood flow was still apparent 48 h after a single injection of drug. This FAA-induced vascular shutdown was virtually abolished in tumor-bearing mice pretreated with an antiserum against tumor necrosis factor, while no such effect was observed in controls pretreated with nonimmune serum (t1/2 of 10.8 +/- 1.2 versus 65.6 +/- 8.0 min for controls). Furthermore, in vitro FAA was seen to induce tumor necrosis factor secretion from murine peritoneal cells and splenocytes. These studies suggest that FAA-induced tumor vascular shutdown in the colon 26 tumor is mediated by tumor necrosis factor.
Animals↗