Biomedical subjects

J Timar

Publications and source records attributed to J Timar.

At least 37 records · Page 2Linked to original sources

PubMed1994

Tumor cell-derived 12(S)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction.

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase inhibitor N-benzyl-N-hydroxy-5-phenyl-pentamide. The identity and stereochemistry of tumor cell-derived 12-HETE was substantiated by gas chromatography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesion to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynthesis by tumor cells, which paralleled the tumor cell-induced endothelial cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both increased 12(S)-HETE biosynthesis and tumor cell-induced endothelial cell retraction. Highly metastatic variants of elutriated B16a cells which had been shown to produce large quantities of 12(S)-HETE induced significant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce endothelial cell retraction. These results suggest that 12(S)-HETE synthesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1994

12-lipoxygenases and 12(S)-HETE: role in cancer metastasis.

Arachidonic acid metabolites have been implicated in multiple steps of carcinogenesis. Their role in tumor cell metastasis, the ultimate challenge for the treatment of cancer patients, are however not well-documented. Arachidonic acid is primarily metabolized through three pathways, i.e., cyclooxygenase, lipoxygenase, and P450-dependent monooxygenase. In this review we focus our attention on one specific lipoxygenase, i.e., 12-lipoxygenase, and its potential role in modulating the metastatic process. In mammalian cells there exist three types of 12-lipoxygenases which differ in tissue distribution, preferential substrates, and profile of their metabolites. Most of these 12-lipoxygenases have been cloned and sequenced, and the molecular and biochemical determinants responsible for catalysis of specific substrates characterized. Solid tumor cells express 12-lipoxygenase mRNA, possess 12-lipoxygenase protein, and biosynthesize 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid], as revealed by numerous experimental approaches. The ability of tumor cells to generate 12(S)-HETE is positively correlated to their metastatic potential. A large collection of experimental data suggest that 12(S)-HETE is a crucial intracellular signaling molecule that activates protein kinase C and mediates the biological functions of many growth factors and cytokines such as bFGF, PDGF, EGF, and AMF. 12(S)-HETE plays a pivotal role in multiple steps of the metastatic 'cascade' encompassing tumor cell-vasculature interactions, tumor cell motility, proteolysis, invasion, and angiogenesis. The fact that 12-lipoxygenase is expressed in a wide diversity of tumor cell lines and 12(S)-HETE is a key modulatory molecule in metastasis provides the rationale for targeting these molecules in anti-cancer and anti-metastasis therapeutic protocols.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1993

Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE.

Cellular motility, a prerequisite for metastasis of tumor cells, is affected by a 55-kDa tumor-cell-secreted cytokine which influences the migration of the producing cells and is called autocrine motility factor (AMF). Previous studies indicated that AMF stimulates motility by binding to its receptor, a cell-surface glycoprotein of 78 kDa (gp78), inducing its phosphorylation, activating a pertussis toxin (PT)-sensitive G-protein, and stimulating inositol metabolism. However, the intracellular signaling mechanisms which transduce and regulate the AMF motility response remain largely unknown. 12-(S)-HETE, a lipoxygenase metabolite of arachidonic acid which affects the cytoskeletal architecture of murine melanoma cells, also stimulates cell motility independently of PT-sensitive G-proteins and up-regulates gp78 surface expression. 12-(S)-HETE induces the phosphorylation of gp78 in a manner analogous to AMF and the motility response of these murine melanoma cells to both AMF and 12-(S)-HETE is inhibited by protein kinase C inhibitors. Furthermore, perturbation of the AMF receptor stimulated endogenous biosynthesis of 12(S)HETE. These results suggest the existence of an "autocrine motility cycle" which influences melanoma cell motility by gp78 activation, and production of second messengers which affect the cytoskeletal architecture and expression of the AMF receptor itself.

Animals↗

PubMed1993

PKC mediates 12(S)-HETE-induced cytoskeletal rearrangement in B16a melanoma cells.

The fatty acid 12(S)-HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)-HETE stimulates cytoskeleton-dependent cellular responses such as adhesion and spreading. Analysis of 12(S)-HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin-binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)-HETE. The 12(S)-HETE-induced cytoskeletal alterations were accompanied by centrifugal organelle-translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)-HETE effect indicated a PKC-mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal-associated protein. Optimal effects were obtained after 5 min treatment with 12(S)-HETE at 0.1 microM concentration. 12(S)-HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)-HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton-dependent cell activity such as spreading.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1993

The lipoxygenase metabolite, 12(S)-HETE, induces a protein kinase C-dependent cytoskeletal rearrangement and retraction of microvascular endothelial cells.

We previously reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], induced large vessel endothelial cell (EC) retraction and increased tumor cell adhesion to exposed extracellular matrix (Honn et al., FASEB J. 3, 2285-2293, 1989). Here, we present evidence that 12(S)-HETE induces the retraction of microvascular ECs in a time- and concentration-dependent manner. The EC retraction was observed 15 min after 12(S)-HETE treatment and reached a peak level between 1 and 2 h. The monolayer reformed by 24 h. Silver staining and "gap-FRAP" experiments suggest that 12(S)-HETE altered the normally apposed cell junctions and impaired gap junction-mediated cell-cell communication. It appears that the 12(S)-HETE effect was mediated by cytoskeletal alteration. The first observed alteration in EC cytoskeleton following 12(S)-HETE stimulation is vimentin bundling, followed by the rearrangement and disruption of vinculin-containing adhesion plaques and/or simultaneous redistribution of alpha-actinin and disruption of spectrin. These changes are accompanied by progressive microfilament dissolution. During the same time interval, alpha-actinin is mobilized to the cell periphery at cell "ruffles." However, 12(S)-HETE showed little or no effects on actin-binding proteins filamin and tropomyosin or on microtubules. 12(S)-HETE effects on these cytoskeletal elements were fully reversible by 24 h and appeared to be mediated through enhancing protein phosphorylation. Following 12(S)-HETE (0.1 microM) treatment increased phosphorylation of proteins that comigrated with myosin light chain (20 kDa), actin (42 kDa), and vimentin (57 kDa) were observed. The enhanced phosphorylation of these cytoskeletal proteins was confirmed by 2D gel analysis. The phosphorylation-promoting effect of 12(S)-HETE on cytoskeletal proteins could be totally abolished by calphostin C, partially inhibited by staurosporine, but was not influenced by N-[2-(methylamine)ethyl]-5-isoquinolinesilfonamide dihydrochloride (HS), suggesting that the 12(S)-HETE effect was mediated via protein kinase C. This was further substantiated by quantitative experiments demonstrating that calphostin C, but not H8, inhibited 12(S)-HETE-induced EC retraction.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1992

The lipoxygenase metabolite 12(S)-HETE promotes alpha IIb beta 3 integrin-mediated tumor-cell spreading on fibronectin.

Tumor-cell interaction with the vessel wall during metastasis involves adhesion, induction of endothelial-cell retraction and spreading on the exposed sub-endothelial matrix. The signals for initiation of tumor-cell spreading and the receptors involved are unknown. A protocol was developed to distinguish between initial tumor-cell (B16 amelanotic melanoma; B16a) adhesion to and spreading on fibronectin. The time for maximum spreading was 50 min. Treatment with a lipoxygenase metabolite of arachidonic acid [12(S)-HETE] resulted in maximum spreading in 15 min (max. effect approx. 0.1 microM). Other lipoxygenase metabolites were ineffective. 12(S)-HETE treatment induced a rearrangement of F-actin, vinculin, vimentin intermediate filaments and integrin alpha IIb beta 3, but not integrin alpha 5 beta 1. Antibodies to alpha IIb beta 3 but not alpha 5 beta 1 blocked the 12(S)-HETE effect on B16a spreading. B16a-cell attachment to fibronectin resulted in increased metabolism of arachidonic acid to 12(S)-HETE, which was inhibited by lipoxygenase but not by cyclo-oxygenase inhibitors. Accordingly, lipoxygenase inhibitors but not cyclo-oxygenase inhibitors blocked spontaneous B16a-cell spreading. The protein-kinase-C inhibitors calphostin C, H7 and staurosporine also inhibited spreading, while the protein-kinase-A inhibitor H8 was ineffective. These data suggest that B16a-cell spreading on fibronectin is initiated by a lipoxygenase metabolite [12(S)-HETE] of arachidonic acid and is mediated by protein kinase C.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1992

Identification of the alpha IIb beta 3 integrin in murine tumor cells.

Previously, we have identified an alpha IIb beta 3-like integrin in tumor cells by using antibodies against platelet alpha IIb beta 3. However, alpha IIb beta 3 was considered to be expressed strictly in megakaryocyte lineage cells. In order to resolve this controversy, the alpha IIb beta 3-like integrin in murine B16 amelanotic melanoma (B16a) cells was characterized at DNA, RNA, and protein levels. The presence of alpha 5, alpha v, alpha IIb, beta 1, and beta 3 genes in B16a cells was confirmed by Southern analysis. mRNAs for all these integrins except alpha v were detectable by Northern blotting. The alpha IIb beta 3 protein was identified by Western blotting using subunit-specific antibodies and by immunoprecipitation using complex-specific antibody. The alpha IIb beta 3 integrin was localized intracellularly by immunocytochemistry. Finally, alpha IIb and beta 3 mRNAs were amplified by reverse transcription-polymerase chain reaction and the identity of alpha IIb was verified by sequencing. Partial DNA and deduced amino acid sequence analysis showed that B16a alpha IIb shares approximately 80% homology with the human alpha IIb and approximately 90% homology with the rat alpha IIb, whereas B16a alpha IIb shares only approximately 26% homology with the human alpha v. These experiments indicate that the alpha IIb beta 3-like protein in B16a cells is the authentic alpha IIb beta 3 and demonstrate, for the first time, that integrin alpha IIb beta 3 is not confined to megakaryocyte lineage cells.

Amino Acid Sequence↗

PubMed1992

Protein-kinase-C inhibitor calphostin C reduces B16 amelanotic melanoma cell adhesion to endothelium and lung colonization.

We recently reported that the Ca(2+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), was involved in rat Walker carcinosarcoma cell adhesion to large-vessel endothelium. We extended our studies to explore the role of this kinase in the adhesion to small-vessel endothelium and lung colonization of murine B16 amelanotic melanoma (B16a). Subpopulations of B16a cells, which differ in lung-colonization potentials, were isolated by centrifugal elutriation from solid tumors. In this study, we demonstrate that cells from a high metastatic sub-population (HM340), when compared with cells from a low metastatic sub-population (LM180), exhibit elevated levels of total cellular as well as membrane-bound PKC. The increase in PKC in cells from the HM340 correlates positively to their increased ability to adhere to murine pulmonary-microvessel endothelial-cell monolayer, and to form pulmonary colonies in syngeneic mice. Calphostin C, a potent and selective PKC inhibitor, decreases in a dose-dependent manner the adhesion to endothelium and the lung colonization of cells from both the low and the high metastatic sub-populations with IC50 at sub-micromolar concentrations. In conclusion, our results suggest that PKC may be a key element in regulating tumor-cell metastasis and that PKC inhibitors may be anti-metastatic agents.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1992

Increased expression of alpha IIb beta 3 integrin in subpopulations of murine melanoma cells with high lung-colonizing ability.

Four subpopulations of B16 amelanotic melanoma cells, possessing different abilities to induce platelet aggregation (TCIPA) and to form lung colonies, were isolated by centrifugal elutriation. The expression of alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 integrins was examined in the 4 subpopulations in order to determine the relationship between integrin receptor expression and tumor-cell metastatic potential. The mRNA of alpha IIb, alpha 5, beta 1 and beta 3 was detectable in the 4 subpopulations by Northern blotting. A gradual increase in mRNAs and cell-surface immunoreactivity of the alpha IIb beta 3 receptor, but not in their gene copies, was observed from the low to the high metastatic subpopulations. The ability of tumor cells to adhere to fibronectin and subendothelial matrix (SEM) increased in parallel. In the high metastatic cells, the alpha IIb beta 3 receptors, but not the alpha 5 beta 1 receptors, were localized to focal adhesion plaques. Incubation of the high metastatic cells with alpha IIb beta 3-specific antibodies reduced their matrix adhesion, TCIPA and lung-colonizing abilities. In contrast, in the low met- astatic cells, SEM adhesion and lung-colony formation were not affected by anti-alpha IIb beta 3 antibody treatment. Incubation of either the low or the high metastatic subpopulation with an alpha 5 beta 1-specific antibody had no effect in vitro and showed a slight inhibition of lung colonization in vivo. Our results suggest that several phenotypic characteristics of the enhanced metastatic potential of B16a subpopulations may be mediated by increased expression of alpha IIb beta 3 receptors and that expression of these receptors may be regulated at the transcriptional level.

Blood Platelets↗

PubMed1992

Is there a role for the tumor cell integrin alpha IIb beta 3 and cytoskeleton in tumor cell-platelet interaction?

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the alpha IIb beta 3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface alpha IIb beta 3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against alpha IIb beta 3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of alpha IIb beta 3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with alpha IIb beta 3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1992

Calcium channel blocker treatment of tumor cells induces alterations in the cytoskeleton, mobility of the integrin alpha IIb beta 3 and tumor-cell-induced platelet aggregation.

Calcium channel blockers of the phenylalkylamine (i.e. verapamil), benzothiazepine (i.e. diltiazem) and dihydropyridine (i.e. nifedipine) classes were evaluated for effects on the tumor cell/platelet interactions using Walker 256 carcinosarcoma cells (W256 cells). When W256 cells were pretreated for 15 min with channel blockers at concentrations of 50-200 microM, macroscopic tumor-cell-induced platelet aggregation was inhibited (order of potency; nifedipine greater than diltiazem much greater than verapamil). However, ultrastructural analysis revealed limited, focal platelet aggregates associated with tumor cell plasma membranes of verapamil- and diltiazem-treated cells. There was no evidence of platelet activation or platelet association with the tumor cell membrane in cells pretreated with nifedipine. Walker 256 cells possess the intergrin alpha IIb beta 3. Tumor cell alpha IIb beta 3 was shown to mediate tumor cell/platelet interactions in vitro [Chopra et al. (1988) Cancer Res. 48:3787]. Patching and capping of surface alpha IIb beta 3 were inhibited by nifedipine greater than diltiazem much greater than verapamil. The degree of inhibition of alpha IIb beta 3 receptor mobility parallels the inhibition of tumor-cell-induced platelet aggregation. W256 cells are characterized by a well-developed microfilament and intermediate filament network and by the absence of a distinct microtubular network. Calcium channel blockers had no effect on the low polymerization level of tubulin. However, they induced rearrangement of microfilament stress fibers. Intermediate filaments were also rearranged but to varying degrees. The order of effectiveness for alteration of intermediate filament organization was nifedipine greater than diltiazem while verapamil was ineffective. We propose that the previously reported inhibition of tumor cell/platelet interaction and tumor cell metastasis by calcium channel blockers [Honn et al. (1984) Clin Exp Metastasis 1:61] is due not only to the effects of the Ca2+ channel blockers on platelets, but also to their effect on the tumor cell cytoskeleton resulting in an inhibition of the mobility and function of the alpha IIb beta 3 receptor.

Animals↗

PubMed1992

Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

Animals↗

PubMed1992

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis.

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB4 induced a modest stimulation but LTC4, LTD4, LTE4 as well as LXA4 and LXB4 were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin alpha IIb beta 3 and monoclonal antibodies against this integrin but not against alpha 5 beta 1, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.

Animals↗

PubMed1992

Development and characterization of a sex-dependent metastatic preputial gland adenocarcinoma in human tumor-bearing immunosuppressed F344 rats.

Newborn F344 rats were treated with polyclonal rabbit anti-thymocyte serum, which caused partial immunosuppression for a limited period. These rats were injected s.c. with cells from human tumor cell lines (two melanomas and a B lymphoma). Within 46 days after the tumor regression a renal carcinosarcoma, resembling Wilms' tumor, developed in each animal, however in male rats an adenocarcinoma of the preputial gland arised. Chromosome analysis and transplantation experiments proved the rat origin of the new tumors. In the present study we describe the biological and morphological characteristics of the adenocarcinoma of the preputial gland (ACPG). Their invasive properties were demonstrated in the lung colony assay, after intra-arterial injection and in the spleen-liver model. ACPG showed unique metastatic phenotype: dependence on the sex of the host.

Adenocarcinoma↗

PubMed1992

Differential expression of proteoglycans on the surface of human melanoma cells characterized by altered experimental metastatic potential.

Heparan sulphate (HS) and chondroitin sulphate (CS) proteoglycans (PGs) frequently have opposite biologic functions in cell-matrix adhesion as well as in the regulation of cell proliferation. Data revealed that sulphated glycosaminoglycans (sGAGs) (sugar chains of PGs) are differently expressed in tumor cells characterized by different metastatic potential; the more metastatic cells contain a higher HS/CS ratio. As the proliferative capacity of tumor cells is also frequently altered in parallel with their metastatic potential, it was not clear whether observed PG alterations reflect changes in cell proliferation or metastatic potential. The cell-associated PG expression and sGAG biosynthesis was studied in tumor cells of human melanoma lines characterized by different experimental metastatic potential to the mouse liver but similar in vitro/in vivo proliferation rates. Using antibodies against PGs we found different expression of PG epitopes in melanoma lines, except from the melanoma antigen. Unlike the low CSPG (melCSPG) metastatic melanoma cells, the cell line with high metastatic capacity contained a higher proportion of positive cells for surface-HSPG without the coexpression of certain cartilage-type CSPG epitopes (recognized by MAb HSFPG 529) as well as by an increased pericellular HS/CS ratio due to intracellular accumulation/retention of CS. Immunocytochemistry of adherent cells revealed HSPGs at substrate-attached membrane areas only in cases of highly metastatic melanoma cells. These data further support our view that the absolute or relative dominance of HSPGs over CSPGs at the cell surface of metastatic tumor cells can be considered a marker of a more metastatic phenotype.

Cell Membrane↗

PubMed1992

[The value of adjuvant irradiation in lymphatic vessel invasion in patients with a cervical carcinoma in histopathological stage Ib and with negative lymph nodes].

The present retrospective study attempts to evaluate the significance of adjuvant radiotherapy as a prognostic factor for stage Ib cervical carcinoma without lymph node metastases but with lymph-vascular space invasion. 54 patients fulfilled the inclusion criteria: histopathological stage Ib, radical hysterectomy with pelvic lymphadenectomy, negative pelvic lymph nodes and lymph-vascular space involvement. Though larger tumors were found (tumor infiltration of the cervix greater than 2/3) in the irradiated group (chi 2-test p = 0.003), there was no significant difference in the five-year overall survival (92% five-year survival without vs. 90% with adjuvant irradiation). In contrast statistical significant difference was found in recurrence-free interval between these groups (Wilcoxon test p = 0.02).

Carcinoma↗

PubMed1991

The lipoxygenase metabolite 12(S)-HETE induces a cytoskeleton-dependent increase in surface expression of integrin alpha IIb beta 3 on melanoma cells.

Integrin receptors are mediators of cell-extracellular matrix and cell-cell interactions. Biochemical and immunocytochemical evidence shows that the platelet integrin receptor alpha IIb beta 3 is present on the cell surface, at focal adhesion plaques and in the perinuclear region of metastatic B16a murine melanoma cells. Antibody to the fibronectin receptor alpha 5 beta i, inhibits basal adhesion by approx. 30%, whereas antibodies to alpha IIb beta 3 are ineffective. The surface immunoreactivity of tumor cells for alpha IIb beta 3 can be enhanced by pre-treatment (5 min) with a lipoxygenase metabolite of arachidonic acid [i.e. 12-(S)-HETE] in a dose-dependent manner (max. effect approx. 0.1 microM). Other lipoxygenase metabolites are ineffective. B16a cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following 12-(S)-HETE pretreatment. This pre-treatment of tumor cells enhances their adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 receptors. 12-(S)-HETE also facilitates the redistribution of alpha IIb beta 3 in the plasma membrane with localization at the focal adhesion plaques. The cytoskeleton of the B16a cell is characterized by an absence of distinct microtubules in interphase cells and the presence of prominent microfilaments and vimentin intermediate filaments. In B16a cells, the disruption of intermediate filaments and/or microfilaments prevents the 12-(S)-HETE-induced increase in plasma membrane alpha IIb beta 3 and enhanced tumor-cell adhesion to fibronectin. The microtubule-disrupting agent, colchicine, is ineffective in both respects. We conclude that the lipoxygenase metabolite of arachidonic acid, 12-(S)-HETE, regulates the surface expression and function of the alpha IIb beta 3 integrin in B16a cells. Further, these data support the hypothesis that microfilaments and intermediate filaments have a profound role in regulating the expression of a multifunctional integrin in B16a tumor cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1991

Localization of receptors for luteinizing hormone-releasing hormone in pancreatic and mammary cancer cells.

Previous work showed that hamster and human pancreatic tumors but not normal pancreata exhibit low-affinity cell-membrane receptors for luteinizing hormone-releasing hormone (LHRH). Although the regression of experimental pancreatic cancers induced by treatment with LHRH agonists or antagonists could be explained in part by the creation of sex-steroid deficiency, direct effects mediated by LHRH receptors might also play a role. Here, we demonstrate that pancreatic tumor cells do exhibit high-affinity binding sites for LHRH, but only in their nuclei; low-affinity sites are associated with the cell membranes. These binding sites appear to be LHRH receptors since electron microscopic immunohistochemical studies show that an antibody to the LHRH receptor reacted with sites in the nucleus of pancreatic tumor cells. Immunoreactive sites in the nucleus also were found in a restricted set of normal hamster pituitary cells thought to be luteinizing hormone-secreting cells and in MXT mouse mammary tumor cells. Such nuclear receptors may be involved in the transmission of the direct action of LHRH analogues on the tumor cells, resulting in the enhancement of programmed cell death.

Animals↗