Effect of transfection with a gene coding for the fibronectin FNIII/10 fragment upon contact inhibition of C3H10T1/2 fibroblasts.

The density-dependent growth inhibition of non-transformed cells may be associated with inefficient transduction of the proliferative signal from cell adhesion molecules. To verify this concept, the C3H10T1/2 fibroblasts were stably transfected with the gene coding for the fibronectin fragment III/10 (FNIII/10). This resulted in differences in gene's expression between original C3H10T1/2 cells and their FNIII/10 transfectants. No significant differences in growth properties were observed in the original or in the transfected cells. C3H10T1/2 cells and their transfectants, when co-cultured, displayed more cells at confluence than the cells cultured alone. Moreover, co-cultured C3H10T1/2 cells and their transfectants showed elevated levels of phospho-ERK1/2 compared to homogenous cultures. Results obtained indicate that cellular homogeneity is responsible for density-dependent growth inhibition.

Modulation of lung tumor colony formation by a subcutaneously growing tumor.

When monodisperse tumor cell suspensions of the mouse sarcoma L1 were injected iv into inbred BALB/c mice bearing subcutaneous grafts of the same tumor, the number of tumor nodules developing in the lungs was highest in animals bearing the oldest primary tumor (28 days) and lowest in animals bearing the oldest primary tumor (28 days) and lowest in animals bearing 16-day-old primary tumors. The number of metastases developing in mice bearing 9-day-old subcutaneous tumors was the same as that in control animals without a subcutaneous tumor. This state of low and high susceptibility to lung metastases can be transferred from tumor-bearing mice to whole-body irradiated mice by means of spleen cells, and this transfer of susceptibility is stable for at least 3 weeks. The results indicate a) that a growing subcutaneous tumor can modulate the number of artificial metastases occurring in the lungs as a result of the iv injection of tumor cells, and b) that this modulation occurs by immune mechanisms. In the model presented here, the host is first protected and then made more susceptible to artificial lung metastases by the presence of a progressively growing subcutaneous tumor.

Stimulatory and inhibitory activities of lung-conditioned medium on the growth of normal and neoplastic cells in vitro.

Lung-conditioned medium (LCM) was obtained by incubation of BALB/c mouse lung tissue fragments in serum-free Eagle medium for 48 hours and subsequent separation by dialysis or chromatography on a Sephadex G-75 column. LCM fractions were tested for their ability to modulate proliferation of normal human endothelial cells and neoplastic cells (N2a, MCF, HEp-2) in vitro as assessed by plating efficiency and tritiated thymidine incorporation assays. It was found that LCM contained two kinds of factors, either stimulating (molecular weight: 50,000-70,000) or inhibiting (molecular weight: 12,000-20,000 and 3,000-5,000) cell proliferation.

Protein kinase C translocation in relation to proliferative state of C3H 10T1/2 cells.

Protein kinase C (PKC) activity of cytosol and membrane fractions of 10T1/2 cells was studied. In cytosol of fast growing cells PKC activity was found in material eluted with 0.150 M NaCl whereas in membrane fractions activity was eluted with 0.065 and 0.150 M NaCl. In the membrane fraction of confluent cells, in contrast to cytosol, very low PKC activity was observed. The translocation pattern of PKC activity eluted with 0.065 M NaCl may be associated with proliferation.

The role of protein kinase C in migration of rat glioma cells from spheroid cultures.

The role of protein kinase C in migration of tumor cells from spheroid cultures was investigated using parental rat glioma cells and their TPA resistant counterparts. These two lines differed in their PKC content as judged by the histone phosphorylation method. Also 4 days of treatment with IRA led to PKC down-regulation. Cells having a drastically decreased PKC level migrated better than those having a normal PKC content.

The pleiotropic effect of TPA on in vitro invasion/migration of glioma and melanoma cell lines.

The invasion/migration of two cell lines, melanoma (MEW) and glioma (BT5C), and their counterparts treated for prolonged time with TPA were studied. On Western blots both cell lines expressed alpha, beta I protein kinase C isoforms, whereas beta II and gamma were not detected. The down-regulation of alpha and beta I PKC was observed in glioma cells after long treatment with TPA, whereas the same treatment of melanoma cells did not lead to PKC downregulation. The down-regulation of PKC was accompanied by stimulation of cell migration/invasion through Transwell chambers coated with collagen IV or fibronectin. In the case of melanoma cells treated with TPA, whose PKC was not down-regulated, the inhibition of migration/invasion was observed. The invasive properties of studied cells did not correlate with any conventional PKC isoform expression.

The effect of phorbol ester treatment on migration of C3H 10T1/2 and BT5C glioma cells: possible application to carcinogenesis.

Treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the second-stage promoter 12-O-retinoylphorbol 13-acetate induced down-regulation of protein kinase C (PKC) and enhanced the migration of C3H 10T1/2 cells. In the case of BT5C glioma cells the same negative correlation was observed only after treatment with TPA. The negative control 4 alpha-phorbol affected neither PKC activity nor the migratory ability of both cell lines.

Peptide analog of fibronectin that inhibits cell migration and ERK 1/2 activity.

Two analogs of the peptide mimicking the 1977-1991 C- terminal part of fibronectin have been synthesized and tested. AWLI simulated human fibronectin fragment 1977-1991, whereas AWLII hybridized to both RGD and 1977-1991 fragments. AWLI and AWLII peptides inhibited the migration of the ovarian carcinoma cell line OVP10 regardless of the presence RGD. AWLI peptide inhibited spontaneous and fibronectin-activated cell migration and ERK1/2 activity. Neither AWLI nor fibronectin induced changes in FAK proteins, as could be judged from Western blots. In conclusion, it seems that the C-terminal fragment of fibronectin inhibits ERK1/2-dependent (random) migration of ovarian carcinoma cells.

The effect of cyclophosphamide on artificial metastases formation in the lungs of Balb/c mice.

Cyclophosphamide (CY) administration prior to intravenous tumor cells injection resulted in a rapid increase of the number of neoplastic nodules in the lungs. It was tested whether the elimination of the possible suppressive effect of CY on the immunological system by reconstitution of the animals with the spleen lymphocytes would diminish CY effect and reduce the number of tumor nodules in the lungs. This procedure only partially eliminates the effect of the growing number of artificial metastases.

Growth of tumor colonies mediated by phorbol ester and 13-cis retinoic acid in mixed cultures.

The aim of the study was to investigate the influence of the substances modifying cell phenotype (phorbol ester and 13-cis retinoic acid) on the growth of mouse neuroblastoma N2a cells in the mixed culture system. The results were compared with monoculture system and with tumor multicellular spheroids system where neoplastic cells are cultivated in the close contact from all sides. The suppression of the pleyotypic effect of phorbol ester was observed when N2a cells were cocultured with 3T3 fibroblasts. On the contrary, the close contact of neoplastic cells to each other (spheroids) do not change the stimulatory effect of phorbol ester on tumor cells proliferation. 13-cis retinoic acid suppressed neoplastic cells growth in all cell culture systems we used.

Mouse neuroblastoma cloning efficiency modulation by quiescent 3T3 cells in culture.

In the mixed culture system nontransformed 3T3 fibroblasts cells inhibited the plating efficiency of neuroblastoma (N2a) cells, whereas heat or methanol killed fibroblasts did not. In contrast, chloroform/methanol extracted 3T3 cells inhibited the plating efficiency of neuroblastoma cells to the same extent as had been the case with living 3T3 cells. In the present study, we discuss the possibility that nontransformed cells surface components can modify the proliferation of neuroblastoma cells.

Growth arrest and polyploidization induced by metahalone microtubule inhibitors on rat glioma cells in culture.

Two pyrimidine analogs (metahalones) NY 3163, NY 3170 have been tested for their effects on the growth of rat glioma cells in monolayer and in spheroid culture. Both substances have earlier been found to inhibit the microtubule system in malignant cells. In glioma cells, arrest of mitosis was accompanied by repeated cycles of DNA synthesis, leading to different levels of polyploidization up to 16 and 32 ploid cells. The effect was not reversible through a culture period of 4 days. Reduced ability of directional migration of cells on a plastic surface was seen for 16 days after exposure. The reaction to microtubule inhibitors seems to differ depending on the type of tumour cells, where some malignant cells have earlier been reported to escape mitotic arrest and proceed to the next G1 phase, in contrast to the present glioma cells which undergo polyploidization.

Indirect effect of cyclophosphamide on the lung tumor colony formation and on the conditioned media activity.

Mice pretreated with cyclophosphamide (CY) had more tumor colonies in lungs following i.v. injection than their intact counterparts. CY treated mice restored with spleen cells showed incomplete reduction of the artificial metastasis in lungs. Conditioned media prepared from lungs contain two factors: stimulating and inhibiting cell proliferation in vitro. Conditioned media from CY treated mice were found to have a decreased inhibitory ability.

Effect of 13-cis-retinoic acid on the spontaneous thymic lymphoma development in AKR mice.

The aim of the study was to analyze the incidence of spontaneous thymic lymphomas in AKR mice kept on a diet with normal and excess retinoid content. The mice whose diet was supplemented with 13-cis-retinoic acid (250 mg per kg chow) developed less lymphomas than those kept on a standard diet (15 mg per kg chow). The effect of cyclophosphamide on the early stage of lymphomogenesis was tested using a single dose (100 mg per kg body weight), injected intraperitoneally to AKR mice. Increased incidence of lymphoma following cyclophosphamide administration was observed as result of a) low sensitivity of prelymphoma and lymphoma cells and/or b) immunosuppressive effect of cyclophosphamide.

The effect of protein kinase C inhibitors on invasion of human ovary cancer cells.

In the present work we tested whether invasiveness of ovarian carcinoma cells could be considered as protein kinase C (PKC) dependent process. The migration and invasion studies were performed in Transwell chambers. Staurosporine, sphingosine and tamoxifen were used as PKC inhibitors. Also the effect of prolonged treatment with TPA was the subject of observation. The obtained results indicated that invasion understood as three step process (attachment, migration and matrix degradation) was affected by PKC inhibitors. The detailed studies, however, showed that attachment and matrix degradation ability of ovarian cancer cells was not changed by PKC inhibitors as opposed to migration which was, at least partly, regulated by protein kinase C.

Protective effect of 13-cis-retinoic acid on hematoporphyrin derivative-induced cell photosensitivity in vitro.

The influence of 13-cis-retinoic acid on the light-caused injury of hematoporphyrin-pretreated nontransformed 10 T1/2 fibroblasts and N2a neuroblastoma cells was investigated. Retinoid reduced the percentage of damaged cells as assessed by the plating efficiency assay. N2a neuroblastoma cells were found to be better protected than 10 T1/2 cells by 13-cis-retinoic acid on hematoporphyrin light-induced injury.

In vitro and in vivo studies of murine sarcoma cells after prolonged treatment with promoting phorbol ester TPA.

Murine sarcoma cell line (L-1) treated with promoting phorbol ester (TPA) showed decreased content and activity of protein kinase C (PKC) as measured by Western blotting and histone phosphorylation methods. The PKC depleted line (L-1R) produced bigger, tumors after s.c. transplantation into syngeneic mice and more spontaneous and artificial metastases developing after i.v. injection of tumor cells. The in vitro studies revealed decreased: adhesiveness, migratory and invasiveness properties of PKC depleted cells. Negative correlation between in vitro and in vivo studies were found.

Action of retinoids during transformation of 10T1/2 cells.

The effect of three retinoids, 13-cis-retinoic acid, arotinoid ethyl sulfone and retinal acetate, on methylcholanthrene (MCA) induced transformation of 10T1/2 cells was studied. It appears that 13-cis-retinoic acid and arotinoid ethyl sulfone prevent transformation in a direct way at the last stage of carcinogenesis. Retinal acetate, however, requires cell-to-cell contacts to inhibit the transformation of 10T1/2 cells.

Phorbol binding and enzymatic activity of protein kinase C from C3H 10T1/2 cells and relation to contact inhibition.

Protein kinase C (PKC) from membrane and cytosol fractions of fast growing as well as confluent contact-inhibited cells was assayed by two methods: PDBu binding and histone phosphorylation. In cytosol and membrane fractions of fast growing cells and in the cytosol fraction of confluent cells a reasonable agreement between the two methods was found. In membranes of confluent cells good PDBu binding was not accompanied with histone phosphorylation. Also in contact-inhibited cells, serum-stimulated hydrolysis of phosphatidylinositol bisphosphate (PIP2) was found to be reduced.

Promoting phorbol ester-TPA enhances migration of C3H 10T1/2 cells. The role of protein kinase C and its relation to carcinogenesis.

Treatment of 10T1/2 cells with promoting phorbol ester drastically enhanced migration of the studied fibroblasts in a serum-supplemented medium. The same cells when exposed to ionomycin or TPA in a serum-free medium did not show any migration. The addition of 1% of serum induced spontaneous and TPA stimulated migration. Also EGF and PDGF separately or together induced the migration of 10T1/2 cells. Parallel studies of protein kinase C documented low enzymatic activity after treatment with TPA, whereas transcripts of PKC were shown independently of TPA treatment.