Cytoplasmic domain mutants of beta1 integrin, expressed in beta 1-knockout lymphoma cells, have distinct effects on adhesion, invasion and metastasis.

Structural requirements for beta 1 integrin cytoplasmic domain functions in adhesion, migration and signaling have been studied mainly for fibroblasts in vitro. The relevance for beta 1-dependent in vivo migration of lymphoid cells has not been assessed. To study this, we transfected beta 1 mutants into beta 1-deficient double knockout (DKO) ESb lymphoma cells, and tested the capacity of the cells to metastasize to liver and spleen. This was compared to alpha 4 beta 1-dependent invasion into cell monolayers in vitro and Mn2+-induced adhesion to fibronectin. Deletion of the five C-terminal residues or mutation of both threonines T788 and T789 to alanines blocked invasion and metastasis and greatly reduced adhesion, in line with known in vitro effects. However, mutations of the NPXY motif tyrosines had unexpected consequences. A Y783F mutation had no effect at all, but a Y783,795F double mutation strongly reduced Mn2+-induced adhesion, whereas it had limited effects on invasion and metastasis. Furthermore, cells expressing a beta 1 beta 2 chimeric subunit, which contains phenylalanines in the NPXY/F motifs, adhered poorly but invasion and metastasis was fully restored to the same levels as for cells expressing wild-type beta 1. We conclude that part of the functions of the beta 1 cytoplasmic domain that are required for adhesion are not essential for beta 1-dependent invasion and metastasis.

Targeted disruption of the beta1 integrin gene in a lymphoma cell line greatly reduces metastatic capacity.

Integrins have been implicated in tumor metastasis. To investigate this, we generated beta1 integrin-negative double knockout (DKO) mutants of the highly metastatic ESb murine T-lymphoma cell line. The in vivo growth capacity of the mutants, which had lost alpha4beta1 and alpha6beta1 expression, was not altered, but their metastatic capacity was greatly reduced. Tail vein injection of 10(4) ESb and single-knockout cells led to death of all animals within 9-11 days. In contrast, only one-half of the animals injected with 10(4) DKO cells died, but much later, after 20-60 days. The other one-half remained disease-free for up to 100 days. Whereas ESb and single-knockout cells disseminated predominantly to liver and spleen, metastasis of DKO cells to these organs was rare, even after this prolonged period. Instead, skeletal muscles were invaded extensively. Metastatic capacity was largely restored in a DKO clone, which had been transfected with beta1 cDNA and expressed beta1 at similar levels as ESb cells. We conclude that beta1 integrins are essential for efficient liver and spleen colonization by the ESb lymphoma.

Activation of G-proteins with AIF-4 induces LFA-1-mediated adhesion of T-cell hybridoma cells to ICAM-1 by signal pathways that differ from phorbol ester- and manganese-induced adhesion.

T-cell hybridomas metastasize widely, and the extent of dissemination correlates with invasiveness in fibroblast cultures. Previously, we provided evidence that both metastasis and in vitro invasion require activation of LFA-1, induced by G-protein-transduced signals triggered by as yet unidentified factors. We show here that LFA-1-mediated adhesion of TAM2D2 T-cell hybridoma cells to ICAM-1 can in fact be induced by direct activation of G-proteins using AIF-4, to the same extent as by using PMA or Mn2+. We assessed effects of protein kinase C (PKC), tyrosine kinase (TK), PI3-kinase (PI3K), and phospholipase C (PLC) inhibitors. Both AIF-4-induced adhesion and invasion were completely blocked by the TK inhibitor genistein and partially blocked by the PI3K inhibitor wortmannin, but not influenced by PKC inhibitor GF109203X. Downregulation of PKC did not affect invasion or adhesion induced by AIF-4 either. In contrast, GF109203X and PKC downregulation blocked PMA-induced adhesion, but genistein and wortmannin had no effect. Invasion and both AIF-4- and PMA-induced adhesion were completely blocked by the PLC inhibitor U73122. Mn(2+)-induced adhesion, which was not or was only partially blocked by the other inhibitors, was delayed by U73122, and spreading of Mn(2+)-treated cells was completely prevented by U73122. However, PLC activity during adhesion was not detected. We conclude that signals required for invasion and G-protein-induced adhesion are similar and are distinct from PKC-induced adhesion, and that in all cases PLC is likely to be activated, but is probably too local and/or transient to be detected.

Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity.

T-cell hybridomas are highly metastatic, and their in vitro invasiveness correlates with metastatic capacity. Invasion is blocked by pertussis toxin (PT), which adenosine diphosphate (ADP)-ribosylates G1-proteins, and we have provided evidence that the PT-sensitive signal stimulates leukocyte function-associated antigen-1 (LFA-1)-mediated adhesion required for invasion. PT pretreatment of TAM2D2 T-cell hybridoma cells reduced metastasis, but only to a limited extent. In the present study, we have transfected the cDNA of the PT ADP-ribosyltransferase S1 subunit into TAM2D2 cells to abrogate G1-protein function permanently. We report here a substantial reduction in the metastatic capacity of two transfectants, S05 and S09, in which 88% and 95% of the G1-proteins was ADP-ribosylated. Two-thirds of the mice injected with S09 cells were tumor-free. Metastasis to the liver was almost completely prevented and less metastases were formed in the spleen and kidneys. Metastasis formation by S05 cells in liver and spleen was much reduced, but in lymph nodes and peritoneal tissues, metastases occurred with a frequency similar to that of controls. We conclude that G1-proteins play an important role in T-cell hybridoma metastasis. We propose that the reduction in metastasis is due to diminished entry of tumor cells from the blood into tissues.

Omental milky spots in the local immune response in the peritoneal cavity of rats.

BACKGROUND: Milky spots have been described as reactive structures, their classification varying from inflamed or haematopoietic tissue to lymphoid organs. In this study we investigated the reactivity of the milky spots in the omentum of rats upon induction of a chronic immune response in the peritoneal cavity. METHODS: At different time points after intraperitoneal administration of Bacillus Calmette-Guérin (BCG), a peritoneal lavage was made, and the omentum and the draining parathymic lymph nodes were taken out. The cellular composition of these tissues was examined on the light microscopic level, using a panel of monoclonal antibodies, and also by electron microscopy. RESULTS: During the first 4 months after administering BCG, the number and size of the milky spots increased enormously. Separate macrophage, T, and B cell areas were formed, but interdigitating cells and follicular dendritic cells were not observed. The number of cells in the peritoneal cavity also increased, and the cellular composition showed a strong similarity with that of the milky spots. Especially during the onset of the experiment, most bacteria were observed in the macrophages in the milky spots rather than in the draining lymph nodes. A cellular immune response was observed in the parathymic lymph nodes but not in the milky spots. CONCLUSIONS: Milky spots, either unstimulated or stimulated, should be classified as perivascular infiltrates. They play a role in the initial clearance of bacteria from the peritoneal cavity. Although the large increase in cell number is predominantly caused by immigration of cells, the results do support the role of milky spots as a site for local proliferation and maturation of especially macrophages and also B cells. The obtained data, however, do not support the earlier made assumption that milky spots function as a secondary lymphoid organ in the peritoneal cavity.

Targeted disruption of CD44 in MDAY-D2 lymphosarcoma cells has no effect on subcutaneous growth or metastatic capacity.

CD44 splice variants have been shown to be involved in metastasis of carcinomas. In addition, the standard form of CD44 has been implicated in metastasis, particularly of melanomas and lymphomas. To investigate this, we have generated a CD44-negative mutant of the highly metastatic murine MDAY-D2 lymphosarcoma. The two CD44 alleles of this diploid cell line were sequentially disrupted by homologous recombination, using isogenic CD44 genomic constructs interrupted by a neomycin or hygromycin resistance-conferring gene. The resulting double knockout (DKO) cells had completely lost the capacity to bind to immobilized hyaluronic acid, but did not differ from MDAY-D2 cells in integrin expression or in vitro growth. Subcutaneous (s.c.) growth potential and metastatic capacity of MDAY-D2 and DKO cells were assessed by s.c. and i.v. injection of the lowest cell dose (10(3) or 10(4), respectively) that gave rise to tumor formation by MDAY-D2 cells in approximately 100% of the mice. Quite unexpectedly, we observed no difference at all in either s.c. growth rate or local invasion into surrounding tissues between MDAY-D2 cells and the CD44-negative DKO cells. Also hematogenous metastasis formation upon i.v. injection was similar: both parental and DKO cells metastasized extensively to the spleen, liver, and bone marrow. We conclude that, at least for these MDAY-D2 lymphosarcoma cells, the standard form of CD44 is dispensable for tumor growth and metastasis. Our results show that targeted disruption of genes in tumor cells is a feasible approach to study their role in tumorigenesis and metastasis.

Adhesion of lymphoma cells to fibronectin: differential use of alpha 4 beta 1 and alpha 5 beta 1 integrins and stimulation by the 9EG7 mAb against the murine beta 1 integrin subunit.

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor alpha 4 beta 1, and MDAY-D2 cells in addition also alpha 5 beta 1. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn2+. In ESb cells, this adhesion was mediated by alpha 4 beta 1. In MDAY-D2 cells, however, only alpha 5 beta 1 was involved, despite alpha 4 beta 1 levels similar to ESb cells. The alpha 4 beta 1 integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An alpha 5 beta 1-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by alpha 4 beta 1. These results indicate that alpha 4 beta 1 function in these cells is suppressed in the presence of alpha 5 beta 1. Adhesion of ESb cells to hepatocytes was inhibited by anti-alpha 4 antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that alpha 4 beta 1 and alpha 5 beta 1 function is not activated during this interaction. The 9EG7 antibody against mouse beta 1 integrin was described to inhibit beta 1 integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that 9EG7 stimulated beta 1-integrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.