Differential regulation of cyclooxygenase-2 in nontransformed and ras-transformed intestinal epithelial cells

To determine signaling pathways responsible for modulation of COX-2 expression in nontransformed and transformed epithelial cells, we studied a rat intestinal epithelial (RIE) cell line expressing constitutively active Ras and RhoA. Expression of COX-2 protein was higher in RIE-RhoA(63L) (four-fold) and RIE-Ras(12V) (seven-fold) cells than in parental cells. Prior work suggests that Ras hyperactivity induces the expression of transforming growth factor (TGF)beta and increases epidermal growth factor (EGF)-related peptide signaling-possible mechanisms for increased COX-2 expression. Expression of COX-2 was stimulated by TGFbeta and TGFalpha in RIE and RIE-Rho(63L) cells, but not further stimulated in RIE-Ras(12V) cells. PD153035, an inhibitor of EGF receptor tyrosine kinase, and PD98059, an inhibitor of Erk, attenuated COX-2 expression in RIE and RIE-RhoA(63L). However, the high levels of COX-2 expression in RIE-Ras(12V) cells were not inhibited by either compound. Titration with a pan-neutralizing anti-TGFbeta antibody did not decrease COX-2 in RIE-Ras(12V) cells, even with concurrent EGFR inhibition. Thus, stimulation of the EGF receptor is important in the modulation of COX-2 expression in nontransformed RIE and RIE-RhoA(63L) cells. In Ras-transformed cells, signaling by additional Ras effector pathways, perhaps the RhoA pathway, must be invoked. Identification of these pathways is critical for therapeutic manipulation of COX-2 expression.     

Differential expression of members of the tumor necrosis factor alpha-related apoptosis-inducing ligand pathway in prostate cancer cells

Androgen ablation therapy induces apoptosis only in androgen-sensitive prostate cancer cells; therefore, other cytotoxic drugs are being used to induce apoptosis in androgen-refractory cells. Mifepristone, an antiprogestin used individually or together with the antiestrogen Tamoxifen, has been recommended for induction of cell death and treatment of several hormonal cancers. However, little is known about the mechanism of action of these drugs in prostate cancer. Therefore, we investigated the effect of Mifepristone on the tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) pathway, a newly identified and very effective member of tumor necrosis factor-alpha family. Mifepristone and Tamoxifen induced significant expression of death receptors in prostate cancer cells in vitro and in xenografts. However, Mifepristone in combination with Tamoxifen did not increase prostate cancer cell death compared with their individual values. The involvement of the TRAIL pathway was further confirmed by the activation of caspase-8 in Mifepristone-treated cells. This was followed by truncation of Bid, confirming that Mifepristone activates the TRAIL pathway. This knowledge is being used to design a combination treatment of TRAIL and Mifepristone to induce significant apoptosis in prostate cancer cells.     

Production of tumor necrosis factor-alpha by normal and malignant B lymphocytes in response to interferon-alpha, interferon-gamma and interleukin-4.

Since autocrine stimulation by tumor necrosis factor-alpha (TNF alpha) may be implicated in the proliferation of normal and malignant B cells, we measured the production of TNF alpha protein by these cells in response to various B-cell stimulatory agents. Purified malignant and non-malignant B lymphocytes were incubated with interleukin-4 (IL4), interferon-alpha (IFN alpha) and IFN gamma, and the supernatants were tested for the production of TNF alpha using an enzyme-linked immunosorbent assay (ELISA). Chronic lymphocytic (CLL) and prolymphocytic (PLL) leukemia cells produced low amounts of TNF alpha, irrespectively of the addition of inducers. Normal B lymphocytes (tonsillar and blood) produced TNF alpha, but the level was not influenced by any of the inducers tested. Hairy cell leukemia (HCL) cells produced TNF alpha in the absence of stimuli and this production was markedly enhanced by addition of IFN alpha or, to a lesser extent, by IFN gamma and IL4. These results contradict the hypothesis that IFN alpha exerts its therapeutic action in HCL by inhibition of autocrine TNF alpha production.     

Cytokine regulation of tumor necrosis factor-alpha and -beta (lymphotoxin)-messenger RNA expression in human peripheral blood mononuclear cells.

The immune response at the molecular level is characterized by a carefully coordinated interplay of both cytokine production and receptor induction. The regulation of these molecules including the closely related tumor necrosis factors alpha (TNF) and beta (lymphotoxin, LT) is still incompletely understood. We have examined the effects of various cytokines on the expression of TNF and LT mRNA in human peripheral blood mononuclear cells (PBMC). Northern blot analysis with total cellular RNA from mixed populations of PBMC revealed that genes coding for TNF and LT were not spontaneously expressed. Treatment of PBMC with recombinant interleukin (IL)-2 resulted in a high level expression of TNF and LT mRNA. Whereas IL-1 beta was equally effective as IL-2 in inducing both TNF and LT mRNA, granulocyte-macrophage colony-stimulating factor selectively induced only TNF mRNA. Both TNF and LT mRNA were minimally induced by IL-1 alpha, IL-3, interferon (IFN)-alpha, or IFN-gamma. Similarly TNF alone had little effect on induction of TNF and LT mRNA. In conjunction with IL-2, cytokines such as IFN-alpha, IFN-gamma, or TNF did not interfere with IL-2 induction of TNF and LT mRNA. Interestingly, IL-4 in combination with IL-2 inhibited the IL-2-driven induction of TNF and LT mRNA. This inhibitory effect of IL-4 was also observed at the level of TNF and LT protein secretion. Furthermore, IL-4 was also inhibitory of IL-2-mediated induction of Tac mRNA in PBMC. These results extend the interrelationship of cytokine regulation of TNF and LT expression. In particular, they reveal the previously unrecognized function of IL-4 in antagonizing the IL-2 induction of TNF, LT, and Tac mRNA in PBMC.     

Shape-dependent regulation of proliferation in normal and malignant human cells and its alteration by interferon

The relationship between cell morphology, proliferation, and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes by plating onto plastic surfaces coated with poly(2-hydroxyethyl methacrylate). Poly(2-hydroxyethyl methacrylate) can precisely vary the shape of cells without toxicity. Cell proliferation was quantitated by cell counts and labeling indices were determined by autoradiography. The normal JHU-1 foreskin fibroblasts and IMR-90 lung fibroblasts exhibited contact-inhibited growth with a saturation density of 2.9 X 10(5) and 2.0 X 10(5) cells/cm2, respectively. These cells also exhibited stringent dependency on cell shape with a mitotic index of less than 3% at poly(2-hydroxyethyl methacrylate) concentrations at which the cells were rounded versus a labeling index of 75-90% when the cells were flat. The malignant bladder carcinoma line RT-4 exhibited partial contact-inhibited growth. Its dependency on cell shape was less stringent than that of normal cells with a mitotic index of 37-40% when rounded and 79% when flat. The malignant fibrosarcoma line, HT1080, was not contact inhibited and was entirely shape independent with a mitotic index of 70-90% regardless of cell shape. Treatment of HT1080 cells with low concentration of human fibroblast interferon (less than 40 units/ml) restored shape-dependent proliferation while having little effect on normal cells. Subantiproliferative doses of interferon were also shown to restore contact-inhibited proliferation control to malignant cells previously lacking it. The concordant restoration of contact inhibition and shape-dependent proliferation in malignant cells by interferon suggest that these two types of proliferation controls represent a manifestation of common regulatory mechanisms. However, since these effects occurred at interferon concentrations below that required to produce an antiproliferative effect, these actions of interferon may be distinct from the antiproliferative action.     

Tumor necrosis factor-A expression in human primary malignant melanoma and its relationship to tumor infiltration by CD3+ cells

Optimal conditions for immunohistochemical staining of tumor necrosis factor-A (TNF-A) in paraffin-embedded tissue sections were established to investigate TNF-A expression in human primary malignant melanomas. Seventeen malignant melanomas of the nodular (NMM) and superficially spreading (SSM) subtypes were analyzed. Twelve of these were TNF-A⁺, while 5 did not stain for the cytokine. To evaluate how TNF-A expression affected the immune response to the tumors, infiltration by CD3⁺ and mac387⁺ cells was investigated in NMM. TNF-A expression seemed to selectively affect the capability of T cells to infiltrate the tumors since TNF-A⁺ tumors were found to have significantly lower levels of infiltrating CD3⁺ cells, while there was no difference in numbers of mac387⁺ cells. These results demonstrate that TNF-A is variably expressed in primary malignant melanoma in vivo and that the T-cell response to TNF-A-expressing NMM is inhibited. © 1996 Wiley-Liss, Inc.     

The inflammatory cytokine tumor necrosis factor-alpha regulates chemokine receptor expression on ovarian cancer cells

Epithelial ovarian cancer cells express the chemokine receptor, CXCR4, which may be associated with increased survival and metastatic potential, but the regulation of this receptor is not understood. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is found in ovarian cancer biopsies and is associated with increased tumor grade. In this report, we show that CXCR4 expression on human epithelial ovarian cancer cells is associated with, and can be modulated by, TNF-alpha. Ovarian cancer cells with high endogenous expression of TNF-alpha expressed higher levels of CXCR4 mRNA and protein than cells with low TNF-alpha expression. Stimulation of ovarian cancer cell lines and primary epithelial cancer cells with TNF-alpha resulted in increased CXCR4 mRNA and protein. The TNF-alpha-stimulated increase in CXCR4 mRNA was due partly to de novo synthesis, and up-regulation of CXCR4 cell surface protein increased migration to the CXCR4 ligand CXCL12. CXCR4 mRNA and protein was down-regulated by anti-TNF-alpha antibody or by targeting TNF-alpha mRNA using RNAi. TNF-alpha stimulation activated components of the nuclear factor kappaB pathway, and overexpression of the inhibitor of kappaB also reduced CXCR4 expression. Coculture of macrophages with ovarian cancer cells also resulted in cancer cell up-regulation of CXCR4 mRNA in a TNF-alpha-dependent manner. Finally, there was a correlation between the levels of TNF-alpha and CXCR4 mRNA in clinical biopsies of ovarian cancer, and TNF-alpha protein was expressed in CXCR4-positive tumor cells. TNF-alpha is a critical mediator of tumor promotion in a number of experimental cancers. Our data suggest that one mechanism may be through nuclear factor kappaB-dependent induction of CXCR4.     

Differential expression of TGFbeta-stimulated clone 22 in normal prostate and prostate cancer

The transforming growth factor-beta (TGFbeta) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFbeta superfamily and functionally related genes between patient-matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFbeta-stimulated clone-22 (TSC-22) and Id4. Verification of their mRNA expression by real-time PCR in patient-matched NP and PC bulk tissue, in laser-captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC-22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC-22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC-22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC-22 protein expression as shown by immunoblotting. TSC-22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC-22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC-22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC-22 warrants further investigation on its role in prostate carcinogenesis and on the TSC-22 pathway as a candidate therapeutic target in PC.     

Modulation of the growth of transformed cells by human tumor necrosis factor-alpha and interferon-gamma

Recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) inhibited growth of the cervical carcinoma cell line, ME-180neo, at doses greater than 50 units/ml, but stimulated the growth of these cells at low doses (0.1-10 units/ml). ME-180neo variants selected for resistance to the cytotoxic effects of rHuTNF-alpha retained the ability to be growth stimulated at all concentrations tested. ME-180neo cells and the rHuTNF-alpha-resistant ME-180neo variants possessed equivalent steady state numbers of TNF-alpha receptors with similar Kd values. Recombinant human interferon-gamma (rHuIFN-gamma) augmented the rHuTNF-alpha-induced cytotoxic response of ME-180neo cells and overcame the resistance of the ME-180neo variants to rHuTNF-alpha cytotoxicity. In separate experiments we were able to show that the number of TNF-alpha binding sites on both rHuTNF-alpha-sensitive and -resistant ME-180neo cells was similar and was increased by treatment with rHuIFN-gamma. These results suggest that the growth stimulation of tumor cells mediated by rHuTNF-alpha can be dissociated from the cytotoxic response and that these responses are not related to the number or affinity of TNF-alpha binding sites.     

Differential EphA2 epitope display on normal versus malignant cells

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer, and in particular, unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties, we have begun to target EphA2 on tumor cells using agonistic antibodies, which mimic the consequences of ligand binding. In our present study, we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells, which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand, as well as this subset of EphA2 antibodies. Finally, we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together, these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells.     

Enhancement of antiproliferative effects of interleukin-1beta and tumor necrosis factor-alpha on human prostate cancer LNCaP cells by coculture with normal fibroblasts through secreted interleukin-6.

The cell-cell interactions between tumor cells and stromal cells are considered to be important in the regulation of tumor development at primary and metastatic secondary sites. We studied the effects of various cytokines on the cell-cell interactions between androgen-dependent LNCaP or androgen-independent PC-3 human prostate cancer cell lines and normal fibroblasts using a co-culture system. Among the tested combinations of cytokines and fibroblasts, strong modulations of cytokine actions were seen in coculture with human normal fibroblasts WI-38. While interleukin (IL)-1beta or tumor necrosis factor-alpha (TNF-alpha) partially suppressed LNCaP cell growth in monoculture, each cytokine completely inhibited it in the case of coculture with WI-38 cells. On the other hand, they did not inhibit PC-3 cell growth significantly, regardless of monoculture or coculture. Conditioned medium prepared from WI-38 cells pretreated with IL-1beta or TNF-alpha also strongly inhibited LNCaP cell growth. In the conditioned medium, marked IL-6 secretion was induced from WI-38 cells by IL-1beta or TNF-alpha. Furthermore, neutralizing antibodies to IL-6 or IL-6 receptor abrogated the antiproliferative effects of IL-1beta- and TNF-alpha-pretreated WI-38 conditioned medium. These results demonstrate that the antiproliferative effects of IL-1beta and TNF-alpha on prostate cancer cells are enhanced by coculture with normal fibroblasts through some diffusible factor(s), such as IL-6, from the stimulated fibroblasts.     

Chimeric form of tumor necrosis factor-alpha has enhanced surface expression and antitumor activity

Tumor necrosis factor (TNF)-alpha is a type-II transmembrane protein that is cleaved by TNF-alpha-converting enzyme (TACE/ADAM-17) to release soluble TNF, a cytokine with potent antitumor properties whose use in clinical applications is limited by its severe systemic toxicity. We found that human cells transfected with vectors encoding TNF without the TACE cleavage site (DeltaTACE-TNF) still released functional cytokine at substantial levels that varied between transfected cell lines of different tissue types. Vectors encoding membrane-associated domains of CD154, another TNF-family protein, conjoined with the carboxyl-terminal domain of TNF, directed higher-level surface expression of a functional TNF that, in contrast to DeltaTACE-TNF, was resistant to cleavage in all cell types. Furthermore, adenovirus vectors encoding CD154-TNF had significantly greater in vivo biological activity in inducing regression of established, syngeneic tumors in mice than adenovirus vectors encoding TNF, and lacked toxicity associated with soluble TNF. As such, CD154-TNF is a novel TNF that appears superior for treatment of tumors in which high-level local expression of TNF is desired.     

Role of protein kinase CK2 in the regulation of tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis in prostate cancer cells

Protein kinase CK2 (formerly casein kinase 2 or II) is a ubiquitous and highly conserved protein Ser/Thr kinase that plays diverse roles such as in cell proliferation and apoptosis. With respect to the latter, we originally showed that elevated CK2 could suppress various types of apoptosis in prostate cancer cells; however, the downstream pathways that respond to CK2 for mediating the suppression of apoptosis have not been fully elucidated. Here, we report studies on the role of CK2 in influencing activities associated with tumor necrosis factor-related ligand (TRAIL/Apo2-L)-mediated apoptosis in prostate carcinoma cells. To that end, we show that both androgen-insensitive (PC-3) and androgen-sensitive (ALVA-41) prostate cancer cells are sensitized to TRAIL by chemical inhibition of CK2 using its specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). Furthermore, we have shown that overexpression of CK2alpha using pcDNA6-CK2alpha protected prostatic cancer cells from TRAIL-mediated apoptosis by affecting various activities associated with this process. Thus, overexpression of CK2 resulted in the suppression of TRAIL-induced apoptosis via its effects on the activation of caspases, DNA fragmentation, and downstream cleavage of lamin A. In addition, the overexpression of CK2 blocked the mitochondrial apoptosis machinery engaged by TRAIL. These findings define the important role of CK2 in TRAIL signaling in androgen-sensitive and -insensitive prostatic carcinoma cells. Our data support the potential usefulness of anticancer strategies that may involve the combination of TRAIL and down-regulation of CK2.