RESUMEN
OBJECTIVES: Ability of a cell to survive without adhesion, and to overcome anoikis, is indispensable for malignant cell invasion and metastasis formation. It has previously been shown that TrkB -neutrophin growth factor receptor might be involved in suppression of apoptosis, induced by the lack of adhesion. The aim of our study was to analyse changes in expression of genes and proteins as well as in biological properties of cancer cells cultured without adhesion. A mouse sarcoma, stable, adherent L1 cell line, derived from a spontaneously arisen Balb/c mouse lung tumour, was established in vitro. MATERIALS AND METHODS: L1 cells resistant to anoikis were established by culture of L1 cells without adhesion, followed by selection of clones with elevated expression levels of TrkB protein. Biological characteristics of the cells were studied by migration/invasion tests and colony forming assay. Gene expression analysis was performed by with the aid of cDNA Gene Expression Array and Real-Time PCR. In vivo experiments were conducted in syngeneic Balb/c mice. RESULTS: Significant changes in gene expression, including higher expression level of TrkB, were found in cells that were able to survive without adhesion. Selected TrkB-expressing clones were found to have higher clonogenicity and invasive potential, formed more colonies in mouse lungs, and induced larger tumours, when injected subcutaneously into Balb/c mice. CONCLUSION: Lack of adhesion induced significant changes in the cancer cells' behaviour, which may result from alterations in gene and protein expression levels, including changes in anoikis-connected protein - TrkB.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Metástasis de la Neoplasia/patología , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sarcoma/patología , Animales , Anoicis , Western Blotting , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/genética , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Tirosina Quinasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sarcoma/metabolismo , Ensayo de Tumor de Célula MadreRESUMEN
Accumulating data suggest that cancers contain a fraction of cells called cancer stem cells (CSCs), that may be responsible for upkeep and relapses of disease. In experimental settings, CSCs are regarded as most effective at tumour initiation in in vivo assays. Since the first isolation of cancer stem cells from acute myeloid leukaemia in 1994, cancer stem cells have been identified in human solid tumours and they have also been found in the established cell lines, based on ability of CSCs to form in vitro colonies of a specific morphology, called holoclones. Our study examined the ability of a mouse sarcoma cell line, derived from a lung metastasis of a BALB/c mouse and established as a stably growing line (L1), to produce holoclones in vitro. We aimed to verify a stemness signature of the holoclone cells. The L1 cell line was found to form holoclone colonies in vitro, which were shown to contain a percentage of CSC-like cells. A fraction of the L1 cells was able to repopulate the original cell line, and presented an increased clonogenic and metastatic potential (18th passage). In addition, MTT assay and flow cytometry of the side population fraction revealed that these cells were more resistant to chemotherapeutic drugs than the original cell line, and over-expressed the anti-apoptotic genes, GRP78 and GADD153. We conclude that mouse L1 sarcoma cell line contains CSC-like cells.
Asunto(s)
Células Madre Neoplásicas/metabolismo , Sarcoma/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Técnicas de Cultivo de Célula , Desdiferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , Células Clonales , Resistencia a Antineoplásicos/genética , Chaperón BiP del Retículo Endoplásmico , Citometría de Flujo , Ratones , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Células Madre Neoplásicas/citología , Sarcoma/genéticaRESUMEN
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.
Asunto(s)
Fibronectinas/genética , Transfección , Animales , Secuencia de Bases , Western Blotting , Técnicas de Cocultivo , ADN Complementario , Ratones , Ratones Endogámicos C3H , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genéticaRESUMEN
Either confluence or serum withdrawal may cause growth arrest of cultured non-transformed cells. Here, we compared sparsely populated and confluent C3H10T1/2 cells with and without serum-containing medium. The following proliferation-relevant end points were examined: cell-cycle distribution, Ki-67 antigen presence, the level of the von Hippel-Lindau (VHL) protein, and gene expression, determined using a microarray approach. In sparse/logarithmic cultures, the fraction of cells in G(0)/G(1) phase increased from 55 to 85% following serum withdrawal. Moreover, the fraction of Ki-67 positive cells dropped from 89 to 47%. In confluent cultures, the majority of cells (80%) were in G(0)/G(1) phase and only 25-30% were Ki-67 positive, regardless of serum presence. In both serum-deprived and contact-inhibited cultures, significant and distinct changes in gene expression were observed. Serum deprivation of sparsely cultured cells resulted in significant over-expression of several transcription factors, while confluent cells showed elevated expression of genes coding for Wnt6, uPar, Tdag51, Egr1, Ini1a and Mor1. These results indicate that contact inhibition and serum withdrawal lead to cellular quiescence through distinct genetic and molecular mechanisms.
Asunto(s)
Inhibición de Contacto/fisiología , Fibroblastos/fisiología , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Inhibición de Contacto/genética , Medio de Cultivo Libre de Suero/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C3H , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
The activities of extracellular signal-regulated kinases (ERK1/ERK2) is required for proliferation of several types of cells. The performed analysis showed stimulation of ERK's by fetal calf serum (FCS) or fibronectin in the C3H 10T1/2 cell cultures at logarithmic phase of growth. The ERKs activity was not stimulated in confluent cells. This could not be accounted for a partial down regulation of ERK since its level was stable in both types of cells regardless of their density and kind of stimulation. Searching for ERK up-stream elements we studied the integrin receptor gene transcript by RT-PCR and focal adhesion kinase (FAK) by Western blotting and phosphorylation assays. It was found that FCS and fibronectin stimulated phosphorylating activity of FAK in the cells at the logarithmic phase of growth, but were inefficient in the confluent cells. RT-PCR showed the presence of alpha5 and beta1 integrin transcripts, and p125FAK was at the same level regardless of the type of stimulation. These data indicate that the ability of FAK to be activated plays an important role in ERK regulation and, in consequence in proliferation and growth inhibition during confluence.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal , Animales , Antígenos CD/metabolismo , Western Blotting , División Celular , Línea Celular , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Integrina alfa5 , Integrina beta1/metabolismo , Ratones , Ratones Endogámicos C3H , Fosforilación , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
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.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Fibronectinas/química , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Western Blotting , Adhesión Celular/efectos de los fármacos , Femenino , Fibronectinas/farmacología , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Neoplasias Ováricas/patología , Fosforilación , Células Tumorales CultivadasRESUMEN
The angiogenic ability of human urothelial cells (HCV-29) and their v-ras and v-raf transfectants was studied. The most pronounced angiogenesis, observed in vivo, induced v-raf-transfected cells. The lowest degree of induction of neovascularization presented cells of the parental line. The increased extent of angiogenesis correlated with the presence of VEGF mRNA as measured by RT-PCR as well as the level of VEGF as visualized by the method of Western blotting.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Genes ras , Linfocinas/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Animales , Línea Celular Transformada , Masculino , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica , Proteínas Oncogénicas v-raf , ARN Mensajero/análisis , Transfección , Urotelio , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Treatment of confluent contact inhibited 10T1/2 cells with TPA or OAG induced a dramatic increase of the number of migrating cells, on cover slides inserted into culture dishes. When cover slides were coated with collagen IV or fibronectin, there was a similar increase of the number of migrating cells. RT PCR showed the presence of alpha PKC gene transcripts and the lack of beta and gamma PKC. Western blot analysis showed translocation of 80 kD alpha PKC to membranous fraction following brief treatment with TPA, and down-regulation of PKC after longer exposure to TPA. Collagen IV and fibronectin treatment of 10T1/2 cells induced MAP kinase, (MEK) kinase in the presence and in absence of FCS. Signal transduction pathway depending on protein kinase C and integrin receptors activation appears to facilitate migration of 10T1/2 cells and may be involved in the mechanism of the escape from contact inhibition of movement.
Asunto(s)
Inhibición de Contacto/fisiología , Quinasa 1 de Quinasa de Quinasa MAP , Proteína Quinasa C/fisiología , Animales , Western Blotting , Carcinógenos/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Transformación Celular Neoplásica , Colágeno/farmacología , Diglicéridos/farmacología , Activación Enzimática/efectos de los fármacos , Fibronectinas/farmacología , Integrinas/metabolismo , Integrinas/fisiología , Ratones , Ratones Endogámicos C3H , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
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.
Asunto(s)
Carcinógenos/farmacología , Sarcoma Experimental/tratamiento farmacológico , Sarcoma Experimental/patología , Acetato de Tetradecanoilforbol/farmacología , Animales , Western Blotting , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Femenino , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Fosforilación , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Sarcoma Experimental/enzimología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Carcinógenos/farmacología , Movimiento Celular/efectos de los fármacos , Isoenzimas/efectos de los fármacos , Invasividad Neoplásica , Proteína Quinasa C/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Regulación hacia Abajo , Glioma/metabolismo , Glioma/patología , Humanos , Isoenzimas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteína Quinasa C/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Glioma/fisiopatología , Ésteres del Forbol/farmacología , Animales , Línea Celular/efectos de los fármacos , Fibroblastos/enzimología , Glioma/enzimología , Glioma/patología , Ratones , Proteína Quinasa C/metabolismo , Ratas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We have examined the effect of protein kinase (PKC) depletion in SV40-transformed Djungarian hamster fibroblasts (DM15 cells) on the level of gap junction permeability. Cx43 electrophoretic mobility, and cell sensitivity to different uncoupling stimuli. After 24 hr exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in DM15 cells was reduced to 20-25% in comparison with intact cells. In PKC-depleted cells the level of dye coupling was 30-40% higher than in the same untreated cultures. Western blot analysis revealed multiple forms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decrease in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA (10(-7) g/ml), mezerein (10(-7) g/ml), teleocidin (10(-8) g/ml), Ca-ionophore A23187 (10(-6) g/ml), insecticide 1,1,1-trichloro-2,2-bis-(p-chlorphenyl)-ethane (DDT) (10(-4) g/ml), and nigericin (10(-5) M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fold decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupling effect of mezerein, teleocidin, and A23187, as well as to new exposure to TPA, and became partially resistant to the effect of DDT. Nigericin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of gap junction permeability and has an important significance as a mediator of the uncoupling effects of such substances as TPA, mezerein, teleocidin, and Ca2+.
Asunto(s)
Transformación Celular Viral/fisiología , Uniones Comunicantes/fisiología , Proteína Quinasa C/fisiología , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Línea Celular Transformada , Cricetinae , Regulación hacia Abajo/fisiología , Electroforesis , Colorantes Fluorescentes , Uniones Comunicantes/efectos de los fármacos , Isoquinolinas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Movimiento Celular , Glioma/fisiopatología , Proteína Quinasa C/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Inhibición de Contacto , Fosfatos de Inositol/metabolismo , Ésteres del Forbol/metabolismo , Proteína Quinasa C/metabolismo , Animales , Células Cultivadas , Fibroblastos/enzimología , Ratones , Ratones Endogámicos C3H , Forbol 12,13-Dibutirato/metabolismo , Acetato de Tetradecanoilforbol/metabolismoRESUMEN
Following addition TNF-alpha to variety of cells the diversity of action has been observed. TNF-alpha induced rapid elevation of intercellular Ca2+ level in all studied cells. This suggests TNF receptor is coupled to phospholipase C.
Asunto(s)
Calcio/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Retinoides/farmacología , Animales , Células Cultivadas , Diterpenos , Metilcolantreno , Ratones , Ésteres de Retinilo , Tretinoina/farmacología , Vitamina A/análogos & derivados , Vitamina A/farmacologíaRESUMEN
Protein kinase C from 10T1/2 cells can be eluted by linear gradient of NaCl in two fractions. Following treatment with 10(-5) M 13-cis-retinoic acid a decrease of total PKC activity was observed, mainly at the expense of the 150 mM NaCl eluted fraction.
Asunto(s)
Isotretinoína/farmacología , Proteína Quinasa C/efectos de los fármacos , Animales , División Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/enzimología , Ratones , Ratones Endogámicos C3HRESUMEN
The aqueous extract from wheat sprouts contains some antimutagenic factor(s). The factor(s) abolish(es) the activity of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in the S9 fraction from Aroclor-treated rat livers and also inhibit(s) the mutagenic activity of benzo[a]pyrene (B(a)P) in the Ames test. The extract (fraction S30) was subjected to initial fractionation by thermal treatment, 3 24-h cycles of dialysis and ultrafiltration. The antigenotoxic activity of fraction S30 amounted to 98% and was unchanged by thermal treatment (100 degrees C, 10 min). Both the dialysate and the dialysis fluid inhibited the mutagenic effect of B(a)P by 48.4 and 48% respectively. The microsomal subfraction inhibited the mutagenicity only in 10%, and the postmicrosomal subfraction in 68%. It is concluded that the extract from wheat sprouts contains at least 2 heat-resistant compounds (or groups of compounds) located within the cell cytosol and showing antimutagenic activity: one group is of low molecular weight and another of high MW. Alternatively, low-molecular compounds could either be free or bound to high-molecular compound(s).
Asunto(s)
Benzo(a)pireno/farmacología , Mutágenos , Extractos Vegetales/farmacología , Salmonella typhimurium/efectos de los fármacos , Animales , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Biotransformación , Microsomas Hepáticos/enzimología , Pruebas de Mutagenicidad , Ratas , Salmonella typhimurium/genética , TriticumRESUMEN
Subcutaneous application of aqueous wheat sprout extract to mice resulted in a slight decrease of the ability of fraction S-9 from their skin to activate DMBA to metabolites mutagenic for S. typhimurium TA 98. Induction by benzo(a)pyrene of sperm abnormalities in mice was diminished after oral administration of the wheat sprout extract; however, even high doses of the extract did not completely abolish the effect of benzo(a)pyrene on spermatozoa. In the carcinogenicity studies, the wheat sprout extract, when applied to mouse skin during the initiation phase, enhanced fourfold the induction of papillomas by DMBA and shortened the period of latency from 9 to 5 weeks.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/antagonistas & inhibidores , Benzopirenos/antagonistas & inhibidores , Extractos Vegetales/farmacología , Triticum , Animales , Carcinógenos/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos BALB C , Mutágenos/antagonistas & inhibidores , Papiloma/inducido químicamente , Salmonella typhimurium/efectos de los fármacos , Espermatozoides/efectos de los fármacosRESUMEN
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.