Rajesh Bazaz

Ectopic expression of platelet integrin αIIbβ3 in tumor cells from various species and histological origin

The integrin αIIbβ3 is a membrane receptor which was considered to be expressed only in cells of megakaryocytic lineage. We have shown that αIIbβ3 is expressed in mouse melanoma B16a cells, and in human prostate adenocarcinoma cells. The purpose of this study was to determine whether the megakaryocytic product αIIbβ3 was functionally expressed in other non‐megakaryocyte lineage tumor cells. By using the reverse transcription polymerase chain reaction (RT‐PCR), we have obtained data demonstrating that αIIbβ3 is expressed in a variety of tumor cell lines (17) derived from different species (human, rat and mouse) and of different histological origins (skin, blood, lung, liver, kidney, cervix, colon, bladder, breast and prostate). Immunostaining of tumor cells with a monoclonal antibody (MAb) to αIIbβ3 demonstrates that αIIbβ3 protein is also expressed in tumor cells. A protein kinase C activator PMA stimulates adhesion of tumor cells to fibronectin and fibrinogen, and this stimulated adhesion is blocked by a function‐blocking MAb directed to αIIbβ3. Our results indicate that the megakaryocytic gene product αIIbβ3 integrin is widely expressed among tumor cells of non‐megakaryocytic lineage, suggesting that ectopic expression of this integrin may play an important role in tumor progression. Int. J. Cancer 72:642–648, 1997.

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 HODEs. 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.

Expression and function of the high affinity αIibβ3 integrin in murine melanoma cells

In resting platelets integrin αIibβ3 is constitutively expressed in an inactive state and it does not recognize soluble proteins. Platelet activation results in a conformational change of the low-affinity αIibβ3 to a high-affinity state which then recognizes plasma fibrinogen. The ectopic expression of αIibβ3 integrin in rodent and human cells derived from solid tumors is well documented, although little is known about its affinity state in these tumor cells. In this study we analysed expression and function of high-affinity αIibβ3 in murine metastatic melanoma B16a cells by using a mAb that specifically recognizes high-affinity αIibβ3 (PAC-1). These tumor cells while in suspension bound PAC-1 and fibrinogen. Immunofluorescent studies of B16a cells indicated that high-affinity αIibβ3 is associated with the Golgi complex and the cell surface. Stimulation of B16a cells with a PKC-activator, 12(S)-HETE, induced translocation of the high-affinity integrin from an intracellular pool to the plasma membrane, which resulted in increased tumor cell adhesion to fibronectin. In addition to participating in 12(S)-HETE-stimulated adhesion of B16a cells, the high-affinity aIIbb3 inte-grin is also involved in tumor cell invasion through a reconstituted basement membrane. In conclusion, results from this study suggest that in non-megakaryocytic lineage B16a cells αIibβ3 is constitutively expressed in a high-affinity state, and that this conformation participates in tumor cell adhesion and invasion.

Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.

12-(S)-HETE Induces Cytoskeleton Phosphorylations and Rearrangement in Melanoma Cells

Lipoxygenase metabolites of arachidonic acid (AA) are responsible for alterations in membrane receptor expression, regulated secretion of cellular products as well as in cell motility (1). These alterations involve extensive cytoskeletal rearrangements as translocations of cytoplasmic proteins stored in transport-organelles are mediated by microtubules or microfilaments (2,3). Intermediate filaments are considered as structural components of the cytoskeleton without active involvement in cytoskeletal rearrangement (4). The levels of phosphorylation of individual cytoskeletal proteins affect their conformation and interaction with other cytoskeletal proteins (s). Therefore kinases (PTK, PKC, PKA or MLCK) play an important role. 12-(S)-HETE was shown to induce cytoskeleton-dependent integrin upregulation in tumor cells (6,7) resulting in an increased matrix adhesion as well as in endothelial cell retraction (8). More recently it was reported that 12-(S)-HETE induces PKC activation and translocation to the plasma membrane (9). Here we provide morphological and biochemical evidences for the effect of 12-(S)-HETE on the tumor cell cytoskeleton.

Fatty Acid Modulation of Cancer Cell Spreading and Cytoskeleton Rearrangement

Tumor cell — extracellular matrix interactions consist of attachment, spreading, migration and digestion of matrix ligands. These interactions are critical determinants during metastasis (1,2) and are mediated by receptors consisting of integrins, cell adhesion molecules (CAM) and proteoglycans (3,4). The first step in matrix interaction is a passive phase characterized by initial ligand/receptor interactions, while the second step, the spreading process, is more complex and includes redistribution of matrix receptors, activation of the signal transduction pathways and rearrangement of the cytoskeleton (5). These events are prerequisites for subsequent tumor cell migration and matrix lysis. The regulatory mechanism(s) of cell spreading is not known. However, those mechanisms which control matrix receptor expression, e.g., TGFβ and other cytokines (i.g., IL-I, TNF) may be involved (6). Previous studies indicated that a 12-LOX metabolite of arachidonic acid (AA), 12-(S)-HETE, serves as a positive regulator of integrin αllbβ3 expression and function in murine tumor cells (7,8). However, it was not clear in these studies which phase (i.e., attachment or spreading) of tumor cell matrix interaction was affected by 12-(S)- HETE. Therefore we have analyzed the effects of 12-(S)-HETE on the spreading of tumor cells on a defined matrix protein, fibronectin (FN).