An essential part for Rho-associated kinase in the transcellular invasion of tumor cells.

Adhesion of tumor cells to host cell layers and subsequent transcellular migration are pivotal steps in cancer invasion and metastasis. The small GTPase Rho controls cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility. Cultured rat MM1 hepatoma cells migrate through a mesothelial cell monolayer in vitro in a serum-dependent, Rho-mediated manners. Among several proteins isolated as putative target molecules of Rho, the ROCK (ROK) family of Rho-associated serine-threonine protein kinases are thought to participate in the induction of focal adhesions and stress fibers in cultured cells, and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin. Transfection of MM1 cells with cDNA encoding a dominant active mutant of ROCK conferred invasive activity independently of serum and Rho. In contrast, expression of a dominant negative, kinase-defective ROCK mutant substantially attenuated the invasive phenotype. A specific ROCK inhibitor (Y-27632) blocked both Rho-mediated activation of actomyosin and invasive activity of these cells. Furthermore, continuous delivery of this inhibitor using osmotic pumps considerably reduced the dissemination of MM1 cells implanted into the peritoneal cavity of syngeneic rats. These results indicate that ROCK plays an essential part in tumor cell invasion, and demonstrate its potential as a therapeutic target for the prevention of cancer invasion and metastasis.

A novel protein that participates in nonself discrimination of malignant cells by homologous complement.

The human complement (C) system protects an individual against substances of nonself origin, including xenografts and microbial pathogens. Human cells express C-regulatory proteins, CD46 and CD55, thereby circumventing attack by C3, a major effector of C. Nevertheless, certain malignant cells, particularly those undergoing apoptotic stress, can activate homologous C, overcoming the regulatory actions of CD46 and/or CD55. The molecular mechanisms whereby malignant cells are tagged by homologous C3 remain largely unknown. We identified a novel gene product that converts human cells into targets for homologous complement. Only malignant cells and cell lines exposed to Fas or X-irradiation stimuli produced this protein, designated M161Ag, which was an unglycosylated 43-kDa protein. Analysis of cloned cDNAs indicated that this molecule was a secretory protein containing five amino acids encoded by TGA codons. Its functions were unique in that once secreted from the tumor cells, it bound back to the surface of these cells and activated homologous complement (C3) via the alternative pathway, allowing for C3 deposition on the membrane. This molecule may offer new insight into innate immunity; surveillance of tumor cells by complement is a common feature in the human immune system.

Complement-mediated tumor cell damage induced by antibodies against membrane cofactor protein (MCP, CD46).

We have developed polyclonal and monoclonal antibodies against human membrane cofactor protein (MCP) to use as tools to investigate the functions of MCP on intact nucleated cells. Two human T cell lines, CEM and TALL, are CR1- and DAF-. Pretreatment of these cell lines with M177 and polyclonal anti-MCP, which inhibit cofactor activity almost completely, resulted in effective C3 deposition immediately following addition of these cells to Mg2+/EGTA/human sera. The deposited C3 remained expressed partly on the cell surface and most of them were gradually converted to C3bi. Some of the deposited C3 were complexed with membrane proteins, since 140- and 250-kD bands became significantly accumulated on SDS-PAGE by treatment with the antibodies. We next tested whether these C3-coated cells were damaged by complement-mediated cytolysis. p18, an inhibitor of membrane attack complex (MAC) formation, was negative in TALL but positive in CEM. TALL was lysed efficiently only by treatment with the polyclonal anti-MCP, while CEM showed only slight lysis with the same treatment. Monoclonal antibodies to MCP, including M177, caused only minimal cell destruction. Based on these results, together with the fact that decay-accelerating factor (DAF) serves as a factor for preventing C3 attack on human cells, we conclude that MCP and DAF cooperatively protect host cells from C3 targeting and, in these T cell lines, MCP is sufficient for preventing C3 deposition even without DAF. After all, human cells undergo almost no autologous complement-mediated cytolysis if they express at least one of the functionally active inhibitors, MCP, DAF, or p18.

Enhancement of metastatic capacity of fibroblast-tumor cell interaction in mice.

A low metastatic clone, G6, was isolated from the B16 melanoma cell line by cloning procedure. When the cells were cultured in vitro with fibroblasts from newborn mice, the lung-colonizing potential of G6 cells was substantially increased. The effect of coculture depended on the number of the fibroblasts. The elevated colonizing potential of G6 cells was reversed to the original low potential by subculturing them for 20 days without the fibroblasts. The culture medium conditioned by G6-fibroblast coculture demonstrated an activity to enhance the lung-colonizing potential of G6 cells, whereas the medium from the culture of fibroblasts alone showed only a little activity. The growth rate and plating efficiency of G6 cells cultured with the fibroblasts or in the conditioned medium did not differ from those of uncocultured G6 cells. The potentiating activity in the conditioned medium was nondialyzable and stable to heating at 80 degrees C for 10 min, but was lost after heating for 10 min at 120 degrees C, or by the treatment with trypsin. These results indicate that the enhancement of lung-colonizing potential of G6 cells could be mediated by a soluble factor(s) released from cocultured fibroblasts.

Factor from rat liver with antiinvasive potential on rat ascites hepatoma cells.

An acid/ethanol extract of normal rat liver, both in vitro and in vivo, inhibited the invasion by a highly invasive subpopulation of rat ascites hepatoma cells, AH 130 (LC-AH cells). The addition of 10-80 micrograms/ml extract inhibited the formation of penetrated colonies of LC-AH cells underneath the cultured mesothelial cell (M-cell) monolayer. The tumor cells pretreated with the extract showed the diminished colony formation. Preincubation of the extract with plasma membranes prepared from LC-AH cells abolished the effect of the extract, suggesting a binding of the inhibitory entity [tentatively termed as "invasion-inhibiting factor" (IIF)] to the tumor cell surface. The extract did not inhibit the growth of LC-AH cells, but suppressed their directed migration underneath the M-cell monolayer. A concomitant i.p. injection of the extract with LC-AH cells into rats prevented the invasion by tumor cells of the peritoneum and formation of tumor nodules in the peritoneum and mediastinum, indicating that IIF inhibited the tumor cell invasion and metastasis in vivo, as well. Upon ultrafiltration and gel fractionation, about 60% of IIF activity was recovered in the fraction corresponding to the molecular weight in the range of Mr 3000-4000. This activity was heat-stable at 100 degrees C at neutral pH but labile at acidic pH and was inactivated by the treatment with pronase. The rest of the activity of IIF was found in the fraction of more than Mr 25,000.

Macrophage potentiation of invasive capacity of rat ascites hepatoma cells.

In vitro and in vivo invasive capacities of rat ascites hepatoma cells (AH 130) that had been cultured on the feeder layers of rat macrophages were examined. The in vitro invasive capacity of the tumor cells was measured by their ability to form tumor cell colonies underneath cultured mesothelial cell monolayers; in vivo invasive capacity was examined by the implantation of the tumor cells into the rat peritoneal cavity. When the tumor cells were precultured on a macrophage feeder layer, the in vitro invasive capacity of the tumor cells increased almost 10 times as much as that of uncocultred control cells. The cocultured tumor cells, when implanted in rat peritoneal cavity, infiltrated extensively in the peritoneum and formed many tumor nodules and enlarged metastatic lymph nodes. Implantation of the uncocultured tumor cells did not develop any macroscopically detectable nodules. The effect of macrophages was reversed by subculturing the cocultured tumor cells without macrophages. Treatment of the tumor cells with the medium conditioned by macrophage culture did not result in the increase in invasive capacity. Almost 50% of the macrophage-mediated enhancement of the in vitro invasive capacity was inhibited by the simultaneous addition of superoxide dismutase and catalase at the time of tumor cell-macrophage coculture.

Potentiation of invasive capacity of rat ascites hepatoma cells by adriamycin.

The effect of Adriamycin on the invasive capacity of rat ascites hepatoma cells, W1, was studied. The invasive capacity of W1 cells was estimated in vitro by counting the number of penetrated single tumor cells and tumor cell colonies formed from the penetrated cells underneath a cultured mesothelial cell monolayer (H. Akedo et al., Cancer Res., 46: 2416-2422, 1986). A considerable increment of the invasive capacity was observed when the tumor cells had been treated with 1.0 to 20.0 microM Adriamycin. This augmentation of invasive capacity of tumor cells was partially inhibited by 60 microM N-acetylcysteine, a scavenger of free radicals. On the other hand, 60 microM N-acetylcysteine did not impair the cytotoxicity of Adriamycin for W1 cells measured by an in vitro tetrazolium-based colorimetric assay for cytotoxicity.

Tumor invasion-inhibiting factor 2: primary structure and inhibitory effect on invasion in vitro and pulmonary metastasis of tumor cells.

The primary structure of tumor invasion-inhibiting factor 2 (IIF-2) purified from bovine liver (A. Isoai et al., Jpn. J. Cancer Res., 81:909-914, 1990) was determined. A computer homology search of the National Biomedical Research Foundation data bank revealed that IIF-2 is identical to the carboxyl-terminal region, residue number [69-89], of high mobility group 17 which is a DNA-binding non-histone protein. IIF-2 synthesized by an automated peptide synthesizer showed similar invasion-inhibitory activity as compared with the purified factor, when tested with the monolayer invasion assay system using highly invasive rat ascites tumor cells. When examined with the other in vitro assay systems using a modified Boyden chamber, the synthetic IIF-2 suppressed the chemotactic migration of highly metastatic B16 melanoma (B16FE7) cells to fibronectin or laminin and invasion through Matrigel. The IIF-2 inhibited neither the cell proliferation nor the binding of cells to fibronectin or Matrigel and also showed no significant inhibition of Mr 90,000 type IV collagenase (gelatinase) obtained from human schwannoma (YST-3) cells. The formation of lung colonies in mice given injections of B16FE7 and Lewis lung carcinoma cells was significantly reduced by the coinjection of the IIF-2. These results suggest that IIF-2 suppresses tumor invasion by impairing cell motility and inhibits the migration of metastasizing cells through extracellular matrix (extravasation steps) following their arrest in the capillary bed of the lung in vivo.

Interaction of rat ascites hepatoma cells with cultured mesothelial cell layers: a model for tumor invasion.

Interactions of rat ascites hepatoma cells with primary cultured layers of rat mesentery-derived cells were studied. The mesentery-derived cells were isolated from rat mesentery and cultured in Eagle's minimum essential medium with a 2-fold concentration of amino acids and vitamins supplemented with 10% calf serum. The primary cultured cells, consisting mainly of mesothelial cells in polygonal shape, forms a "paving stone" sheet. Upon seeding the tumor cells on the mesentery-derived cell layers, three different types of tumor cell growth were observed. Type 1 was the formation of piled-up tumor cell nests on mesothelial cell layers. Type 2 was the formation of flattened tumor cell islands underneath mesothelial cell layers. This island formation was clearly observed under a phase contrast microscope 2 days after the tumor cell seeding. Protrusion of cellular processes of the tumor cells beneath mesothelial cells was occasionally seen. Type 3 was the growth of tumor cells in suspension. These types of tumor cell growth closely resemble those in the peritoneal cavity observed after i.p. implantation of the tumor cells. When the tumor cells recovered from the blood of tumor-bearing rats were seeded, flattened tumor cell islands were formed 15 times more frequently than when the tumor cells isolated from host peritoneal cavity were seeded. Shortly after the appearance of small flattened tumor cell islands, a distinct morphological change of mesothelial cells from polygonal to spindle shape was seen preferentially at the marginal area of the cell layers (a partial retraction of cell edges). The retraction of mesothelial cells was induced not only by seeding the tumor cells but by adding the tumor ascites fluid or the medium conditioned by the tumor cell culture. The morphological change was reversed by changing the culture medium to remove the effectors. These results indicate that the system described in this study can provide a useful model to study tumor cell invasion.

Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors prevent the conversion of HMG-CoA to mevalonate and thereby inhibit the synthesis of other products derived from this metabolite. This includes a number of small prenylated GTPases involved in cell growth, motility, and invasion. We studied the effect of HMG-CoA reductase inhibitors (fluvastatin and lovastatin) on in vitro invasion of human pancreatic cancer PANC-1 cells. Epidermal growth factor (EGF) induced a dose-dependent increase of PANC-1 cell invasion in a modified Boyden chamber assay. Stimulation of cancer cells with EGF induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly. Furthermore, Clostridium botulinum C3 transferase, a specific inhibitor of Rho, inhibited the ability of EGF to promote invasion, indicating that EGF-induced cancer cell invasion is regulated by Rho signaling. Treatment of PANC-1 cells with fluvastatin markedly attenuated EGF-induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly, whereas it did not inhibit the tyrosine phosphorylation of EGF receptor and c-erbB-2. The induction of cancer cell invasion by EGF was inhibited by the addition of fluvastatin or lovastatin in a dose-dependent manner. The effects of fluvastatin or lovastatin on cell morphology and invasion were reversed by the addition of all-trans-geranylgeraniol but not by the addition of all-trans-farnesol. These results suggest that HMG-CoA reductase inhibitors affect RhoA activation by preventing geranylgeranylation, which results in inhibition of EGF-induced invasiveness of human pancreatic cancer cells.

Putrescine-dependent invasive capacity of rat ascites hepatoma cells.

The effects of inhibitors of polyamine synthesis on the invasive capacity of rat ascites hepatoma (LC-AH) cells were examined by in vitro assay of penetration of the LC-AH cells through a monolayer of calf pulmonary arterial endothelial (CPAE) cells. Pretreatment of LC-AH cells with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, before seeding them onto a CPAE cell monolayer and culturing them for 24 h in the absence of DFMO decreased the number of penetrating tumor cells time and dose dependently (about 35% of the maximal inhibition) without affecting their viability or proliferative activity. DFMO treatment caused a marked decrease in the intracellular level of putrescine but not of spermidine or spermine. The DFMO-induced decreases in invasive capacity and putrescine level were almost completely reversed by the addition of putrescine to the medium during pretreatment with DFMO or invasion assay but were not affected by exogenous spermidine or spermine. No change in the invasive capacity was observed when the CPAE cells were treated with DFMO and the LC-AH cells with methylglyoxal-bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, which depressed the spermidine and spermine levels but increased the putrescine level in the LC-AH cells. These results suggest that intracellular putrescine modulates the in vitro invasive capacity of LC-AH cells.

Angiogenesis inhibitor TNP-470 inhibits human breast cancer osteolytic bone metastasis in nude mice through the reduction of bone resorption.

Angiogenesis inhibitor TNP-470, 6-O-(N-chloroacetyl-carbamoyl)-fumagillol, semisynthetic analogue of fumagillin, has strong inhibitory activities against in vivo tumor growth and metastasis in a wide variety of tumors. However, it is still unknown whether this agent inhibits bone metastasis. We examined the effects of TNP-470 in a bone metastasis model in nude mice in which intracardiac injection of the human breast cancer cell line MDA-MB-231 (MDA-231) produced osteolytic bone metastasis. After inoculation of MDA-231 cells into the left heart ventricle, TNP-470 (30 mg/kg, three times a week) or PBS was s.c. administrated for 4 weeks. After this period, the TNP-470 had reduced not only the number and area of osteolytic bone metastases (approximately 60 and 70%, respectively) but also their radiolucency. Histological examination of the femurs of the untreated group revealed that most of the cancellous bone had been replaced by the metastatic cancer. Numerous active osteoclasts were present along the trabecular bone surface surrounded by the metastatic MDA-231 cancer cells aggressively invading the bone marrow. In contrast, in the bone from TNP-470-treated mice, bone destruction was markedly inhibited, and there were much fewer osteoclasts. In a murine bone marrow culture under 1,25-dihydroxyvitamin D3 in which mature functional osteoclasts formed in vitro, TNP-470 significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells. And also, TNP-470 suppressed the in vivo bone resorption in calvaria treated with interleukin-1beta, an osteoclast stimulator. These data suggested that TNP-470 inhibited bone metastasis through not only antitumor action by its angiogenesis inhibition but also by the inhibition of osteoclastic bone resorption. Our results indicate that TNP-470 should be a potentially beneficial drug to be used in the treatment of osteolytic metastasis.

Inhibitory effects of tumor invasion-inhibiting factor 2 and its conjugate on disseminating tumor cells.

Tumor invasion-inhibiting factor 2 (IIF-2) is a polypeptide of 21 amino acids which binds to the surface of tumor cells and inhibits experimental invasion in vitro. An albumin conjugate of IIF-2 was used to examine its potential as an antimetastatic compound. The conjugate inhibits in vivo lung metastasis of various highly metastatic tumor cells, including murine melanoma, colon adenocarcinoma, squamous cell carcinoma, forestomach carcinoma, and human fibrosarcoma. In addition to the anti-lung metastasis activity of this compound, it also showed the inhibitory effects on liver and spleen metastasis of murine T-lymphoma cells. A single administration of the conjugate with melanoma cells resulted in prolonged survival times, and their lung colonization was also inhibited when the conjugate was administrated i.v. at times ranging from 6 h before to 1 h after tumor cell inoculation. Similarly, i.p. administration 1 h prior to melanoma cell injection suppressed lung colonization. Pharmacokinetic analysis revealed that the conjugate was more stable than IIF-2 peptide alone. Approximately 10% of the conjugate remained circulating 2 h postinjection and persisted 20 h without degradation, compared with rapid clearing of the unconjugated IIF-2 peptide within 5 min. Furthermore, spontaneous lung metastasis of murine melanoma and colon adenocarcinoma cell was inhibited by successive i.p. administration of the conjugate before the removal of the primary site, with no effect on primary tumor growth. The conjugate significantly reduced tumor cell arrest in the lung and both the IIF-2 peptide and its conjugate demonstrated potent inhibition of basal as well as cytokine-induced-stimulated tumor cell motility. These results suggest that one mode of IIF-2 action may be inhibition of the extravasation of metastasizing cells which have arrested in a target organ, and that the IIF-2-albumin conjugate may be a potent antimetastatic substance with utility in the prevention of artificial seeding of tumor cells during surgical removal of the primary lesions as well as inhibiting metastasis from established metastases.

Cooperation of fibronectin with lysophosphatidic acid induces motility and transcellular migration of rat ascites hepatoma cells.

We have previously shown that the transcellular migration of rat ascites hepatoma (AH130-MM1) cells through a cultured mesothelial cell monolayer (MCL) is triggered with lysophosphatidic acid (LPA) that stimulates actin polymerization and myosin light chain phosphorylation through the activation of Rho-ROCK (Rho-kinase) cascade. When, however, the motility of MM1 cells on a glass surface was tested by phagokinetic track motility assay, LPA failed to induce the motility. Nevertheless, when the glass had been coated with fibronectin (FN), LPA could induce phagokinetic motility which was accompanied by transformation of MM1 cells to fusiform-shape and assembly of focal adhesion. beta1 integrin, the counter receptor of FN, was expressed on MM1 cells. Anti-FN antibody, anti-beta1 integrin antibody and cyclo-GRGDSPA remarkably suppressed LPA-induced phagokinetic motility. These antibodies suppressed LPA-induced transcellular migration through MCL, as well. These results indicate that actin polymerization and phosphorylation of myosin light chain through Rho activation are insufficient for inducing motility but the cooperative FN/beta1 integrin-mediated adhesion is necessary for both the phagokinetic motility and transcellular migration of MM1 cells.

Synergistic effects of basic fibroblast growth factor and hematopoietic growth factors on colony formation of K562 human leukemic cells.

The effect of basic fibroblast growth factor (bFGF) alone and in combination with other hematopoietic growth factors on the colony formation of K562 human leukemic cells was studied using soft agar colony assay. bFGF was found to have a weak colony-stimulating activity on K562 cells derived from the blastic crisis cells of human chronic myelogenous leukemia and to potentiate the K562 cell colony-stimulating activity of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) at a low concentration of below 1 ng/ml. These findings suggested that bFGF stimulates the growth of human leukemic cells directly in vivo alone and in synergy with other hematopoietic growth factors.

Increased expression of S100A6 (Calcyclin), a calcium-binding protein of the S100 family, in human colorectal adenocarcinomas.

The expression of S100A6 (also known as Calcyclin/2A9/ 5B10/PRA) in surgically resected human colorectal adenocarcinomas was examined to investigate whether S100A6 plays a role in the malignancy of human tumor cells. Western blot analysis using the lysates from colorectal adenocarcinomas and adjacent normal mucosa from 10 patients revealed that the average S100A6 level of adenocarcinomas was significantly higher (about 2.4-fold) than that of normal mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A6 antibody (mAbA6) demonstrated that 2(5%) of 42 normal mucosa and 6 (46%) of 13 adenoma specimens were mAbA6-positive and showed granular staining localized at the supranuclear regions of epithelial cells, whereas 23 (55%) of 42 adenocarcinomas and 13 (100%) of 13 carcinoma cells that metastasized to the liver were mAbA6-positive and showed diffuse cytoplasmic staining. A significant correlation between S100A6 expression and Dukes' tumor stage or lymphatic permeation but not with other clinicopathological factors was shown. S100A6 was stained more intensely in peripheral portions than in central portions of adenocarcinomas, whereas Ki-67 (a growth marker) was stained equally in these two portions. These results suggest that S100A6 may be involved in the progression and invasive process of human colorectal adenocarcinomas.

Initial, continuous and intermittent bolus cardioplegia administration: efficacy of potassium-chloride and magnesium-sulfate as minimal additives for minimally-diluted blood cardioplegia.

AIM: The most effective delivery of blood cardioplegia (BCP) remains controversial, and a combination of initial continuous and intermittent bolus BCP seems to compensate each demerit. However, a large amount of crystalloid solution is infused into the myocardium in this method, which may nullify the advantages of BCP. We examined the hypothesis that minimally-diluted BCP resolves this issue and provides superior myocardial protective effects. METHODS: Seventy patients undergoing elective coronary revascularization between 1997-2001 (M:F=55:15, mean age 67.6+/-7.5 years) were randomly allocated into one of 2 groups: Group C (n=35) was given the standard 4:1-diluted blood-crystalloid BCP, and Group M (n=35) was given minimally-diluted BCP supplemented with potassium-chloride and magnesium-sulfate. The BCP temperature was maintained at 30 degrees C. Cardioplegic arrest was induced with 2 minutes of initial antegrade BCP infusion, followed by continuous retrograde BCP infusion. Intermittent antegrade BCP was infused every 30 minutes for 2 minutes. RESULTS: The time required for achieving cardioplegic arrest was significantly shorter in Group M (47.5+/-16.3 vs 62.5+/-17.6 s, p<0.0001) and the number of patients showing spontaneous heart-beat recovery after aortic unclamping was significantly larger in Group M (28 vs 15, p=0.0029). The number of patients suffering from atrial fibrillation during the postoperative period was significantly smaller in Group M (3 vs 11, p=0.034). The total amount of crystalloid solution infused as cardioplegia was significantly smaller in Group M (62.8+/-22.3 vs 733.6+/-382.6 mL, p<0.0001). Postoperative maximum dopamine dose (3.57+/-2.46 vs 5.44+/-2.23 mg/kg/min, p=0.0014) and peak creatine kinase-MB (19.5+/-8.5 vs 25.8+/-11.9 IU/L, p=0.0128) were significantly lower in Group M. The number of patients showing paradoxical movement of the ventricular septum by early postoperative echocardiography was significantly smaller in Group M (9 vs 24, p<0.0007). CONCLUSIONS: These results demonstrate that initial continuous and intermittent bolus administration of minimally-diluted BCP supplemented with potassium and magnesium can be a simple, reliable and effective technique of intraoperative myocardial protection.

Proteolytic elimination of decay-accelerating factor (DAF): lytic abnormality coincides with removal of DAF in papain-treated human erythrocytes.

Erythrocytes (E) from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack decay-accelerating factor (DAF) and this partly causes increasing susceptibility of the E to complement. Several reagents have been used to convert normal E to the complement-sensitive (PNH-like) cells. The relationship between DAF amounts and complement susceptibility of these PNH-like cels has been examined. Of the reported reagents for preparation of PNH-like cells, 2-amino-ethylisothiouronium bromide (AET), papain, and periodate efficiently converted normal E to the complement-sensitive cells, but only papain reduced the quantity of DAF on the cells. Further, of the proteases we tested only papain cleaved DAF to liberate its major fragment from the cells. The papain-treated cells lysed in a similar fashion to PNH cells as the serum concentration increased. The major papain-digested product of DAF had Mr, 55,000, lacked hydrophobicity, and retained the ability to inhibit the C3 convertases. These findings suggest that papain allows liberation from cells of functional domains as well as most of the antigenic epitopes of DAF to generate a PNH-like cell.

Correlation of alkaline phosphatase activity with cell population density in tissue culture cells originated from rat liver.

Alkaline phosphatase (ALP) activity of cultured Li-10 cells obtained from rat liver was found to be a function of cell population density. After the cells grew to confluence, the enzyme activity per cell increased about 100 times that at a low population density. The increase of activity was inhibited by the addition of actinomycin D or cycloheximide to the culture medium. When the cells that had gained high ALP activity after confluency were subcultured, ALP activity decreased to a low basal level after about 48 h. Under cytochemical examination using an electron microscope, the induced ALP activity was seen exclusively at the apical surface region of the cells but scarcely at cell-cell and cell-substratum contact regions.

Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP-elevating agents via a Rho-dependent pathway.

1-Oleoyl lysophosphatidic acid (LPA) induces transmonolayer migration (in vitro invasion) of rat ascites hepatoma MM1 cells and their morphological changes leading to the migration. We have previously shown that an LPA analog, palmitoyl cyclic phosphatidic acid (Pal-cPA), suppresses transmonolayer migration of MM1 cells by rapidly increasing the intracellular cyclic AMP (cAMP) concentration. We report here that various cAMP-elevating agents, including dibutyryl cAMP, forskolin, cholera toxin and 3-isobutyl-1-methylxanthine, consistently inhibited LPA-induced transmonolayer migration of MM1 cells. Moreover, pull-down assays for GTP-bound, active RhoA demonstrated that the blockage by cAMP-elevating agents of morphological changes leading to the migration was probably mediated through inhibiting RhoA activation.