Cloning and characterization of human ubiquitin-processing protease-43 from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol RaSH

Defects in growth control and differentiation occur frequently in human cancers. In the case of human melanoma cells, treatment with a combination of fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss of proliferative potential and tumorigenic properties with a concomitant induction of terminal differentiation. These changes in cellular properties are associated with an induction and suppression in specific subsets of genes that occur in a temporal manner. To identify the complete repertoire of gene changes occurring during melanoma reversion to a more differentiated state a number of molecular approaches are being used. These include, subtraction hybridization using temporally spaced cDNA libraries, random cDNA isolation and evaluation by reverse Northern blotting and high throughput microarray analysis of subtracted cDNA clones. In the present study we have used a novel approach, rapid subtraction hybridization (RaSH), to identify and clone an additional gene of potential relevance to cancer growth control and terminal cell differentiation. RaSH has identified a human ubiquitin-processing protease gene, HuUBP43, that is differentially expressed in melanoma cells as a function of treatment with IFN-beta or IFN-beta + MEZ. HuUBP43 is a type I interferon inducible gene that is upregulated in a diverse panel of normal and tumor cells when treated with IFN-beta via the JAK/STAT kinase pathway. This gene may contribute to the phenotypic changes induced by IFN-beta during growth arrest and differentiation in human melanoma cells and other cell types as well as the antiviral and growth inhibitory effects of interferon.     

Calcium-independent growth of human ovarian carcinoma cells

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.     

Synergistic interactions of interleukin 1, interferon-beta, and tumor necrosis factor in terminally differentiating a mouse myeloid leukemic cell line (M1). Evidence that interferon-beta is an autocrine differentiating factor

The effect was investigated of combinations of cytokines known to be cytostatic for some tumor cells, namely interleukin 1 alpha (IL-1 alpha), interferon-beta (IFN-beta), and tumor necrosis factor (TNF), on the growth and differentiation of the mouse myeloid leukemic cell line, M1, cells. IL-1 alpha, IFN-beta, and TNF by themselves are antiproliferative for M1 cells. Treatment of cells with a mixture of any two of the three cytokines resulted in at least additive growth inhibition. None of these cytokines by themselves induced differentiation of M1 cells as assessed by increased expression of Fc receptors (FcR), stimulation of phagocytic activity and by morphologic criteria. However, as little as 1 U/ml IL-1 alpha in conjunction with IFN-beta or TNF increased FcR expression, phagocytic activity and morphologic changes in addition to inhibiting the growth of M1 cells. The combination of IFN-beta and TNF did not induce differentiation, although the growth of the cells was markedly inhibited. Both TNF and lipopolysaccharide (LPS) induced the in vitro production of IFN activity by M1 cells. Furthermore, the induction of differentiation of M1 cells by a combination of IL-1 alpha with either IFN-beta, TNF, or LPS was inhibited by antibody against mouse IFN-beta. Therefore, it appears that IFN-beta provides one of the two required signals for differentiation of M1 cells by these combinations of stimulants, the other being IL-1. Furthermore, the cytostatic effect of TNF by itself on M1 cells was also partly blocked by anti-IFN-beta antibody, suggesting that IFN-beta is also involved in the growth inhibitory effect of TNF for M1 cells. In contrast, the cytostatic effect of IL-1 on M1 cells was not blocked by anti-IFN-beta antibody. In conclusion, both the cytostatic and differentiative effect of TNF appear to be mediated by IFN-beta. Thus, the combination of IL-1 and IFN-beta or inducers of IFN-beta resulted in terminal differentiation of M1 cells. Northern blot analysis using cDNAs for murine IFN-beta1 or human IFN-beta2 showed an increased expression of mRNA for IFN-beta1 but not for IFN-beta2 by stimulation with TNF or LPS, strongly suggesting that IFN-beta 1 rather than IFN-beta 2 is responsible for TNF or LPS effects.     

Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cell types, but it is also known as an antimitogenic factor for several types of tumor cell lines. The biological processes by which HGF inhibits tumor cell growth remain poorly understood. Here we report a comparative study of HGF-mediated signal transduction events between two opposite responding types of human hepatoblastoma cell lines, HuH6 and HepG2. Following serum starvation, both cell lines were cultured in hepatocyte growth medium (HGM), a chemically defined medium, in the presence or absence of HGF. Under these culture conditions, cell growth in HuH6 was promoted by HGF, while it was inhibited in HepG2. Phosphorylation of p42/mitogen-activated protein (MAP) kinase was observed within 10 min after HGF stimulation in both cell lines. The level of phosphorylated MAP kinase in HuH6 declined to basal levels after 2 hr. However, in HepG2 the phosphorylated form was detectable at 6 hr. p21/waf1 was induced in both cell lines where levels peaked 4–6 hr after HGF stimulation. In HuH6, a marked decrease of p21/waf1 was observed at 8–12 hr, while a high level of p21/waf1 was sustained for at least 24 hr in HepG2. HGF treatment depressed cdk2 activity in a time-dependent manner in HepG2 while the activity increased in HuH6. When serum-starved HepG2 was growth stimulated with serum in the presence or absence of HGF, the cells treated with HGF underwent growth inhibition correlating with a sustained induction of p21/waf1 and a decrease of cdk2 activity. Immunoprecipitation analysis revealed accumulation of cdk2-associated p21/waf1 in the HGF-treated HepG2. Together, the results suggest that sustained induction of p21/waf1 mediates growth inhibition in HepG2 in the presence of HGF. J. Cell. Physiol. 177:130–136, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

Inhibition of epidermal growth factor receptor activity by retinoic acid in glioma cells

The growth inhibitory effects of exogenously added retinoic acid (RA) on various cultured human glioma cells was observed to be heterogenous, with an ID50 ranging from 10(-7) M to no response. The protein tyrosine kinase activity of epidermal growth factor receptor (EGF-receptor) appeared to parallel the cell's growth responsiveness to RA. Cells sensitive to RA-induced growth inhibition exhibited a dose-dependent decrease in EGF-receptor activity, whereas RA-resistant cells showed no alterations in EGF-receptor protein tyrosine kinase activity or expression. The modulation of EGF-receptor by RA was further examined with RA-sensitive (LG) and -resistant (NG-1) cell lines. Both cell lines were approximately equal in their ability to bind and internalize epidermal growth factor in the presence or absence of RA. Several independent assays suggested that the inhibition of EGF-receptor activity was independent of protein kinase C modulation as mediated by phorbol myristate acetate. However, alterations in associated glycoconjugates of EGF-receptor were observed among the sensitive cells but not the resistant cells. These results suggest RA-induced growth inhibition in sensitive cells may arise, at least in part, through alterations in EGF-receptor and structure.     

l-Leucine induces growth arrest and persistent ERK activation in glioma cells

Glioma is the most common type of brain tumor, and has the worst prognosis in human malignancy. Experimental evidence suggests that the use of high concentrations of various amino acids may perturb neoplastic cell growth. Thus, the aim of this study was to investigate whether essential amino acids can alter the growth and proliferation of glioma cells. Studies were performed using C6 rat glioma cell lines. High concentration of L-leucine induced growth arrest of glioma cell lines. Terminal transferase uridyl nick end labeling assay and cell cycle analysis showed that the effect of L-leucine on glioma cells growth was not cytotoxic, but rather cytostatic. Additionally, the extracellular signal-regulated protein kinase was activated in L-leucine-treated glioma cells, and inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK) enhanced the effect of L-leucine on glioma cell growth. These data suggest that high concentration L-leucine combined with inhibition of MEK is a potential strategy for glioma cell growth arrest.     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

Inhibition of protein kinase C arrests proliferation of human tumors

We have shown that inhibition of protein kinase C by 1-5-isoquinolinylsulfonyl-2-methylpiperazine, H7, induces differentiation and inhibits proliferation of Neuro 2a cells. We have now tested if H7 is able to inhibit proliferation of: 1) human tumor cell lines from tissues other than brain; and 2) primary cultured cells from several human brain tumors. H7 inhibits, in a dose-dependent manner, proliferation of all human tumor cell lines tested and of primary cultured cells from human brain tumors. These results indicate that inhibition of protein kinase C inhibits proliferation of tumoral cells, therefore, H7, and likely other inhibitors of protein kinase C, could be useful in the clinical treatment of brain (and probably other) tumors.     

RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.     

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.     

A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1.

Earlier studies demonstrated the induction of beta 2-interferon (IFN-beta 2) in human diploid fibroblasts (FS-4 strain) exposed to tumor necrosis factor (TNF). These studies suggested that IFN-beta 2 mediates an antiviral effect in TNF-treated cells and exerts a feedback inhibition of the mitogenic effect of TNF. Here we demonstrate that the expression of the antiviral action of TNF can be enhanced by prior exposure of FS-4 cells to trace amounts of IFN-beta 1. IFN-beta 1, at a higher concentration, can directly increase the expression of IFN-beta 2. Exposure of cells to TNF enhanced IFN-beta 2 (but not IFN-beta 1) mRNA expression in response to poly(I).poly(C), an IFN inducer which is also known to stimulate FS-4 cell growth. Platelet-derived growth factor and interleukin-1 also led to the increased expression of IFN-beta 2. However, platelet-derived growth factor and interleukin-1 could override the antiviral effect of TNF and also that of exogenously added IFN-beta 1. Our data suggest that a complex network of interactions that involves the endogenous production of IFN-beta 2 is triggered by several growth-modulatory cytokines. Cellular homeostasis is likely to represent a balance between the induction of IFN-beta 2 by these cytokines and their ability to override the inhibitory actions of IFN-beta 2.     

Differential effects of type I IFN and IFN-gamma on the binding of tumor necrosis factor to receptors in two human cell lines

The effect of IFN-alpha and IFN-beta on the expression of cell surface receptors for tumor necrosis factor (TNF) was examined in two human cell lines. In HeLa cells, IFN-alpha and IFN-beta increased 125I-TNF binding, whereas in HT-29 cells these two IFN either slightly decreased or had no effect on 125I-TNF binding. In contrast, IFN-gamma increased 125I-TNF binding in both cell lines. Both IFN-alpha and IFN-beta exerted an antagonistic effect on IFN-gamma-induced stimulation of TNF receptor expression in HT-29 cells, but did not inhibit TNF receptor induction by IFN-gamma in HeLa cells. IFN-gamma and, to a lesser extent, IFN-beta were synergistic with TNF in producing cytotoxic/cytostatic activity in HT-29 cells. Despite the inhibitory effect of IFN-beta on the IFN-gamma-induced stimulation of TNF receptor expression, IFN-beta did not inhibit the synergistic enhancement of TNF cytotoxicity by IFN-gamma in HT-29 cells. The dissociation between the effects of IFN-beta on TNF receptor expression and on the cytotoxic activity of TNF in HT-29 cells suggests that TNF receptor modulation is not a major mechanism of synergism between IFN and TNF.     

Study of motogenic signal in human melanoma cells

The components of the extracellular matrix (ECM) are more than just adhesion sites for migrating tumor cells: following enzymatic degradation of the ECM, the release of sequestrated growth factors increases, thus they become available for tumor cells. In a number of cancers dysfunction of epidermal growth factor receptor (EGFR) or hepatocyte growth factor receptor (c-Met) contribute to the malignant transformation that directly regulates cell proliferation, survival and motility. Furthermore, intracellular calcium level plays an important role in the regulation of the tyrosine kinase pathway. In our preclinical experiments, by administering heparin-derived oligosaccharides we influenced the interaction between human melanoma cells and ECM. In vitro cell migration was inhibited by heparin fragments. Moreover, two of the effective oligosaccharides reduced the number of lung colonies formed in SCID mice. In human melanoma cells an important element of Ca2+ homeostasis, the purinergic Ca2+ channel P2X7 proved to be an anti-apoptotic protein. EGFR and c-Met showed constitutive activity in human melanoma cells, and their inhibition in vitro caused decreased proliferation, migration and elevated apoptosis. Administration of a selective c-Met-TKI significantly decreased primary tumor growth in vivo as well as the capacity for liver colony formation in SCID mice. Selective EGFR-TKI had less inhibitory effect on metastasis formation, and had no effect on the primary tumor. Our results suggest the necessity of a rational dual-specific drug design for the purpose in the therapy of malignant melanoma.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

1,2-Dioctanoyl-glycerol induces a discrete but transient translocation of protein kinase C as well as the inhibition of MCF-7 cell proliferation

Exposure of MCF-7 human breast cancer cells to phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) results in a dose-dependent inhibition of cell proliferation. One of the earliest biochemical events induced by TPA is the translocation of protein kinase C from the cytosolic to the particulate compartment. We have investigated the effects of permeant diacylglycerol 1,2-dioctanoyl-glycerol (diC8) on both protein kinase C activity and MCF-7 cell proliferation. DiC8 induces a discrete but significant translocation of protein kinase C within the first minutes of MCF-7 cell treatment (26 +/- 6%, mean +/- SD of 5 different experiments, upon 5 min incubation in the presence of 43 micrograms/ml diC8). However, this effect is only transient as the enzymatic activity returns to the control value after 60 min. DiC8 mimics the effect of TPA on MCF-7 cell proliferation. The dose-response curves for both protein kinase C translocation and cell growth inhibition show that diC8 exerts its effects on both parameters in the same range of concentrations, despite some discrepancies at the lowest doses. We also report that long-term treatment of the cells with diC8 does not lead to the protein kinase C disappearance observed during prolonged exposure to TPA. All together, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation and suggest that the enzyme translocation but not its down-regulation could be a pre-requisite in the biological cell response.