Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.     

Changes in cytoarchitecture and mobility in B16F1 melanoma cells induced by 5-Br-2'-dU coincide with Rock2, miRNAs 138-5p and 455-3p reciprocal expressions

ROCK2 is a protein involved in the restructuring of the cytoskeleton in cell adhesion and contractibility processes. miR-138-5p and miR-455-3p regulate Rock2 expression, cell proliferation, migration, and invasion in different experimental cell models. However, their participation in the cytoarchitecture and mobility of B16F1 melanoma cells exposed to 5-Br-2'-dU is partially known. This work aimed to analyze ROCK2 and miRs 138-5p and 455-3p expression associated with morphological and mobility changes of B16F1 mouse melanoma cells exposed to the thymidine analog 5-Bromo-2'-deoxyuridine (5-Br-2'-dU). We observed an increase (2.2X n = 3, p < 0.05) in the cell area, coinciding with an increase in cell diameter (1.27X n = 3, p < 0.05), as well as greater cell granularity, capacity for circularization, adhesion, which was associated with more significant polymerization of F-actin, collapsed in the intermediate filaments of vimentin (VIM), and coinciding with a decrease in migration (87%). Changes coincided with a decrease in Rock2 mRNA expression (2.88X n = 3, p < 0.05), increased vimentin and a reciprocal decrease in miR-138-5p (1.8X), and an increase in miR-455-3p (2.39X). The Rock2 kinase inhibitor Y27632 partially rescued these changes. These results suggest ROCK2 and VIM regulate the morphological and mobility changes of B16 melanoma cells after exposure to 5-Br-2'-dU, and its expression may be reciprocally regulated, at least in part, by miR-138-5p and miR-455-3p.     

Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter's antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA–Raf–MEK–ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC–ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects.     

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH₄Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state.     

The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.     

Adenoviral transduction of melanoma cells with B7-1: antitumor immunity and immunosuppressive factors

 Previous studies in experimental models have demonstrated that the transduction of human or murine melanoma cells with the co-stimulatory B7-1 molecule induces effective antitumor immune responses. In order to develop B7-1 gene transfer as a therapeutic tool in the clinical management of melanoma, efficient means of in vivo gene transfer must be used. To this end we evaluated in vitro and in vivo immune responses associated with adenoviral transduction of murine and human melanoma cells with B7-1. Adenovirus-mediated transduction of human and murine melanoma cells with B7-1 leads to high-level transgene expression in vitro and in vivo and does not affect MHC class I and II expression. Adenovirus-delivered B7-1 induced antitumor immune responses, on the basis of observations that human melanoma cells transduced to express human B7-1 were able to co-stimulate allogeneic and autologous T cells to proliferate and that murine melanoma K1735 cells transduced to express murine B7-1 were rejected by syngeneic, immunocompetent mice. By contrast, intratumoral injection of an adenovirus encoding murine B7-1 failed to eliminate established murine melanoma (K1735) despite high-level transgene expression in tumor cells. Potent T cell inhibitory factor(s) secreted by both K1735 cells and select human melanoma cells may contribute to the failure to achieve protection in this setting. Thus, immune inhibitory melanoma-derived factors need to be taken into account when considering the clinical use of B7-1 immunotherapy. Received: 21 December 1997 / Accepted: 16 March 1998     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

Distinct and non-cross-reactive epitopes are recognized on B16 melanoma by LAK cells and anti-B16 monoclonal antibodies.

Two rat anti-B16 melanoma monoclonal antibodies (MoAb), designated IB16-6 and IB16-8, recognize an epitope expressed with high density on the surface of B16 parental cells and B16-F1, F10, F10FLR, and BL6 sublines. The purpose of this study was to define by means of cytolytic and clonogenic assays whether these MoAbs reacted with the same or distinct determinants as those recognized on B16 targets by lymphokine-activated killer (LAK) cells. Using 125I-labeled antibody and Scatchard analysis, the affinity constant (KA) of IB16-6 was determined to range from 5.6 to 9.4 x 10(8) liter/M and the number of receptor sites per B16 cell was 4.8 x 10(4) to 2.5 x 10(5). The effects of anti-B16 MoAb on LAK activity were determined by either preincubating 51Cr-labeled B16 target cells with varying concentrations of MoAb, followed by the cytolytic assay, or exposing unlabeled B16 cells to MoAb, and then carrying out a 10-day clonogenic assay. Over a wide range of antibody concentrations, IB16-6 and IB16-8 had minimal effects on LAK activity, and even at MoAb concentrations up to 1 mg there were no changes in target cell sensitivity or colony-forming ability. Enzymatic treatment of B16 melanoma cells with either trypsin or pronase completely removed the epitope recognized by MoAb IB16-6 but did not alter B16 sensitivity to LAK cells. These observations indicate that the LAK recognition unit was distinct from the epitope reactive with MoAb IB16-6 and that the B16 determinant(s) recognized by LAK cells is resistant to proteolytic enzymes. The molecular structure of each of these remains to be determined.     

Alpha 1- and beta 2-adrenergic receptors co-expressed on cloned MDCK cells are distinct glycoproteins

We have explored the molecular differences between alpha 1- and beta 2-adrenergic receptors that are co-expressed by a clonally-derived cell line, Madin-Darby canine kidney clone D (MDCK-D). MDCK-D membranes were pre-labeled with selective alpha 1- and beta-adrenergic radioligands and were then solubilized with the non-ionic detergent digitonin. Solubilized alpha 1- and beta 2-adrenergic receptors were retained by immobilized wheat germ agglutinin and were eluted following addition of N-acetyl-D-glucosamine or sialic acid. Both receptors were also retained by immobilized Limax flavus lectin, a sialic acid-binding lectin. Lectins that were specific for N-acetyl-D-glucosamine residues did not bind to these receptors. These results indicate that both alpha 1 and beta 2 receptors are sialylated glycoproteins. The solubilized alpha 1- and beta 2-adrenergic receptors migrated with different elution profiles from an Ultragel AcA 34 column. The apparent molecular sizes of the digitonin-receptor complexes were 68A for the alpha 1 receptor and 55A for the beta 2 receptor. These results show that alpha 1- and beta 2-adrenergic receptors can be present on the same cell as distinct sialic acid-containing glycoproteins.     

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.     

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.     

Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin alpha(5) in promoting melanoma metastasis. The alpha(5) expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin alpha(4), alpha(v), and beta(1) subunits. Neutralization of alpha(5) with anti-alpha(5) antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between alpha(5) and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin alpha(5) is positively correlated with melanoma metastasis and might be an anti-melanoma target.     

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes

 In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-γ release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients. Received: 17 August 2000 / Accepted: 1 February 2001     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Expression of the cell adhesion molecules N-CAM and L1 in B16 melanoma cells

The cell adhesion molecules N-CAM and L1 are important for cell-cell recognition and cell migration and so may be involved in the metastatic process. We have studied the biosynthesis of N-CAM and L1 in the B16 melanoma cell lines B16-F1 and B16-F10 which differ in metastatic capacity. N-CAM was synthesised as two glycosylated polypeptides with Mr of 150,000 and 210,000; L1 was synthesised as one polypeptide with Mr of 215,000. In fetal neurons N-CAM is synthesised as a 135,000 and a 200,000 Mr polypeptide and L1 as a 200,000 Mr polypeptide. Thus, the Mr of N-CAM and L1 in tumour cells appeared to be 10,000-15,000 higher than in the normal cells. L1 was phosphorylated in the tumour cells as in neurons. The tumour cells also phosphorylated the 210,000 Mr N-CAM polypeptide, whereas no phosphorylation of the 150,000 Mr polypeptide was observed. In neuronal cells both the corresponding polypeptides are phosphorylated and thus the biosynthesis of N-CAM in tumour cells seem to differ from that in neuronal cells with regard to phosphorylation. No differences in biosynthesis of N-CAM or L1 were apparent between the two tumour cell lines, B16-F1 and B16-F10.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Effects of trace metals on mouse B16 melanoma cells in culture

The effects of fourteen metal ions (As³⁺, As⁵⁺, Cd²⁺, Co²⁺, Cr³⁺, Cr⁶⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Se⁴⁺, V⁵⁺, VO²⁺) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10⁻⁴ M, metal ions such as As³⁺, As⁵⁺, Cd²⁺, Cr⁶⁺, Se⁴⁺, V⁵⁺, VO²⁺, and, to a minor extent, Li⁺, Hg²⁺, and Co²⁺ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg²⁺>Cr⁶⁺=Cd²⁺>As³⁺, As⁵⁺>V⁵⁺, VO²⁺>Se⁴⁺=Ni²⁺=Co²⁺=Li⁺. An increased synthesis of melanin in B16 cells was noted after incubation with Co²⁺, Ni²⁺, Cd²⁺, and Li⁺, whereas Se⁴⁺ had, on the contrary, an inhibiting effect on melanogenesis.     

Hepatocyte growth factor induces pro-apoptotic genes in HepG2 hepatoma but not in B16-F1 melanoma cells

Hepatocyte growth factor (HGF) exerts a cytostatic effect on HepG2 and B16-F1 cell lines. To evaluate the possible involvement of the apoptotic process in this effect, we performed studies at cellular and molecular levels. HGF induced apoptosis only in HepG2 hepatoma cells at day 3 in about 20% of the cells undergoing growth inhibition, while hallmarks of apoptosis did not occur in B16-F1 melanoma cells. During the first 24 h after HGF treatment, enhanced expression of the pro-apoptotic genes bax and c-Myc was observed at level of mRNA and protein. Concomitant induction of antizyme (AZ) might lower ornithine decarboxylase (ODC) protein level though a huge increase in ODC mRNA level took place. This was suggested as a signal for apoptosis decisional phase. The levels of the proteins examined except that of AZ fell down thereafter when HepG2 cells underwent apoptosis. In B16-F1 cells, only ODC and AZ protein levels were elevated probably in relation to the initial elevated growth rate and the absence of apoptosis involvement in the following cytostatic effect of HGF in melanoma cells. Consistent with this hypothesis, bax mRNA and protein levels were unchanged or even lower relative to control values.