The survival function of the Bcr-Abl oncogene is mediated by Bad-dependent and -independent pathways: roles for phosphatidylinositol 3-kinase and Raf.

The Bcr-Abl tyrosine kinase constitutively activates cytokine signal transduction pathways that stimulate growth and prevent apoptosis in hematopoietic cells. The antiapoptotic action of interleukin-3 (IL-3) has been linked to a signaling pathway which inactivates the proapoptotic protein Bad by phosphorylation through kinases such as Akt and Raf. Here we report also that expression of Bcr-Abl leads to phosphorylation of Bad in hematopoietic cells. Bad phosphorylation induced by Bcr-Abl is kinase dependent, requires phosphatidylinositol 3-kinase (PI3-kinase), and mitochondrial targeting of Raf, and occurs independently of Erk. The ability of Bcr-Abl to confer cytokine-independent survival to hematopoietic cells was compromised by inhibitors of PI3-kinase, as well as by a dominant negative form of Raf targeted to the mitochondria. Furthermore, when the capacity of Bcr-Abl to phosphorylate Bad was completely blocked by dominant negative Raf, a subpopulation of cells remained viable, providing evidence for Bad-independent survival pathways. This alternative survival pathway remained PI3-kinase dependent. Finally, Bcr-Abl, but not IL-3, inhibited the proapoptotic activity of overexpressed Bad. We conclude that the antiapoptotic function of Bcr-Abl is mediated through pathways involving PI3-kinase and Raf and that survival can occur in the absence of Bad phosphorylation.

Growth inhibition of a human colorectal carcinoma cell line by interleukin 1 is associated with enhanced expression of gamma-interferon receptors.

Recombinant human tumor necrosis factor and recombinant human gamma interferon (IFN-gamma) exert synergistic growth inhibitory effects in WiDR human colorectal carcinoma cells. In this cell line, tumor necrosis factor increases IFN-gamma binding. Interleukin 1 (IL-1) is a cytokine that mimics many of the biological actions of TNF. Therefore, in the present study, we investigated the effects of recombinant human IL-1 on cell growth and IFN-gamma receptor expression in WiDR cells. IL-1 slightly inhibited the growth of WiDR cells, and exerted additive growth inhibitory effects in the presence of IFN-gamma. IL-1 caused a time- and dose-dependent increase in 125I-labeled IFN-gamma binding that was maximal at 6 h, persisted for at least 24 h, and was blocked by both actinomycin D and cycloheximide. The increase in binding was associated with an increase in cell surface IFN-gamma receptor protein expression as determined by Scatchard analysis of equilibrium binding data and by immunofluorescent staining with an anti-human IFN-gamma receptor monoclonal antibody. IL-1 also produced a time- and dose-dependent increase in IFN-gamma receptor mRNA levels that was maximal at 3 h and persisted for at least 24 h. Actinomycin D, but not cycloheximide, completely blocked the IL-1-mediated increase in IFN-gamma receptor mRNA levels. However, IL-1 did not alter IFN-gamma receptor mRNA half-life. These data indicate that IL-1 and IFN-gamma exert additive growth inhibitory effects on colon cancer cell growth, and suggest that IL-1 increases IFN-gamma receptor expression in these cells by enhancing IFN-gamma mRNA levels.

Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia.

We identified TMPRSS2 as a gene that is down-regulated in androgen-independent prostate cancer xenograft tissue derived from a bone metastasis. Using specific monoclonal antibodies, we show that the TMPRSS2-encoded serine protease is expressed as a Mr 70,000 full-length form and a cleaved Mr 32,000 protease domain. Mutation of Ser-441 in the catalytic triad shows that the proteolytic cleavage is dependent on catalytic activity, suggesting that it occurs as a result of autocleavage. Mutational analysis reveals the cleavage site to be at Arg-255. A consequence of autocatalytic cleavage is the secretion of the protease domain into the media by TMPRSS2-expressing prostate cancer cells and into the sera of prostate tumor-bearing mice. Immunohistochemical analysis of clinical specimens demonstrates the highest expression of TMPRSS2 at the apical side of prostate and prostate cancer secretory epithelia and within the lumen of the glands. Similar luminal staining was detected in colon cancer samples. Expression was also seen in colon and pancreas, with little to no expression detected in seven additional normal tissues. These data demonstrate that TMPRSS2 is a secreted protease that is highly expressed in prostate and prostate cancer, making it a potential target for cancer therapy and diagnosis.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppress tumor necrosis factor secretion in vitro.

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon-gamma. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells.

The cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-gamma) were studied in five human pancreatic tumor cell lines. During a 48-h incubation, MIA PaCa-2 cells were most sensitive to rhTNF (56% cytotoxicity, 500 U/ml), T3M4 cells were most sensitive to rhIFN-gamma (54% cytostasis, 250 U/ml), and ASPC-1 and COLO 357 cells were most sensitive to the combination of rhTNF and rhIFN-gamma (56 and 55% cytotoxicity, respectively, 250 U/ml of each cytokine). The PANC-1 cells were relatively insensitive to either the individual or the combined effects of these cytokines. All five cell lines exhibited specific, high-affinity receptors for 125I-labeled rhTNF (480-8,610 sites/cell) and rhIFN-gamma (2,050-6,280 sites/cell). The MIA PaCa-2 cells, which were the most sensitive to the inhibitory effects of rhTNF, also possessed the largest number of 125I rhTNF receptors; all other cell lines had a relatively low number of binding sites and low sensitivity. In contrast, no direct correlation could be made between the number of IFN-gamma binding sites and inhibitory sensitivity in any of the cell lines. Incubation of COLO 357 cells at 37 degrees C with either 125I rhTNF or 125I rhINF-gamma led to internalization of the respective 125I-labeled ligand. Our findings document the presence of cytokine receptors in human pancreatic carcinoma cells and suggest that postreceptor events rather than differences in receptor number or affinity more likely govern the responsiveness of pancreatic cancer cells to TNF and IFN-gamma.

Tumor necrosis factor up-regulates gamma-interferon binding in a human carcinoma cell line.

WiDR colorectal carcinoma cells are highly sensitive to the synergistic cytotoxic effects of tumor necrosis factor (TNF) and gamma-interferon (IFN-gamma). In the present study, we have investigated the effects of recombinant human (rh) TNF and IFN-gamma on the binding of both ligands in this cell line. WiDR cells exhibited high affinity binding sites for both 125I-rhTNF (Kd = 1.66 x 10(-10) M, 920 sites/cell) and 125I-rhIFN-gamma (Kd = 4.15 x 10(-10) M, 18,960 sites/cell). Preincubation of the cells with rhTNF (24 h) increased cell-associated 125I-rhIFN-gamma radioactivity by 129% when binding was carried out at 37 degrees C, as a result of an increase in both surface bound and internalized 125I-rhIFN-gamma. However, rhTNF did not alter the degradation profile of released 125I-rhIFN-gamma radioactivity. Scatchard analysis of 125I-rhIFN-gamama binding data (4 degrees C) revealed that rhTNF induced a 245% increase in 125I-rhIFN-gamma binding sites. Conversely, rhIFN-gamma caused a 68% increase in 125I-rhTNF binding sites and a 58% increase in receptor affinity. rhIFN-gamma also increased the subsequent binding of 125I-rhIFN-gamma, whereas rhTNF increased the subsequent binding of 125I-rhTNF. Furthermore, preincubation of the cells with both rhTNF and rhIFN-gamma also resulted in an increase in the binding of both ligands. Actinomycin D and cycloheximide blocked all the effects of rhTNF and rhIFN-gamma on ligand binding. However, the basal level of 125I-rhIFN-gamma binding was insensitive to either inhibitor, whereas the basal level of 125I-rhTNF binding was decreased by both inhibitors. These data indicate that in some cell types TNF and IFN-gamma may induce an increase in their own receptors (homologous up-regulation) and concomitantly increase each other's receptors (heterologous up-regulation) and that these actions are due, in part, to enhanced receptor synthesis.

Upregulation of interferon-gamma binding by tumor necrosis factor and lymphotoxin: disparate potencies of the cytokines and modulation of their effects by phorbol ester.

Tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) are immune-modulating cytokines that exert synergistic cytotoxic effects in several types of tumor cells, including ASPC-1 human pancreatic carcinoma cells. Lymphotoxin (LT), is a cytokine that binds to the TNF receptor and mimicks most of the biological actions of TNF. In the present study, we examined ASPC-1 cells for cytokine-mediated modulation of TNF and IFN-gamma receptors. Treatment of ASPC-1 cells with recombinant human IFN-gamma (rhIFN-gamma) did not significantly alter 125I-rhTNF binding. In contrast, treatment with rhTNF led to a dose- and time-dependent increase in 125I-rhIFN-gamma binding and internalization. Scatchard analysis revealed that rhTNF increased the number of 125I-rhIFN-gamma binding sites from 11,000 sites/cell to 23,000 sites/cell without altering receptor affinity. Although rhLT also increased 125I-rhIFN-gamma binding, it was 100-fold less potent than rhTNF. In contrast, rhLT was only 10-fold less potent than rhTNF in displacing 125I-rhTNF from its receptor. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) blocked the rhLT- and rhTNF-mediated increase in 125I-rhIFN-gamma binding and markedly decreased 125I-rhTNF binding. These data suggest that both TNF and LT upregulate IFN-gamma receptors in ASPC-1 cells, but that LT is much less efficient than TNF. Further, the TPA-induced attenuation of IFN-gamma receptor upregulation suggests that protein kinase C activation can regulate the TNF/LT-mediated pathways involved in IFN-gamma receptor upregulation.

Long-term effect of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells.

The long-term cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-gamma) were studied in five human pancreatic carcinoma cell lines. The pancreatic tumor cell lines were heterogenous in their response to individual cytokines. During a 7-d incubation, MIA PaCa-2 cells were more sensitive to rhTNF than rhIFN-gamma, whereas ASPC-1, T3M4, and COLO 357 cells were more sensitive to rhIFN-gamma than rhTNF. PANC-1 cells were relatively insensitive to both cytokines. In a previous report, we demonstrated synergistic cytotoxic effects of rhTNF and rhIFN-gamma during 48-h incubations in ASPC-1 and COLO 357 cells (Raitano et al, Pancreas, April, 1990). In this study, a 7-d treatment with both rhTNF and rhIFN-gamma did not produce synergistic effects in any of the cell lines. However, a 24-h treatment with rhIFN-gamma, followed by removal of the cytokine, markedly increased the long-term cytotoxic/cytostatic effects of rhTNF in ASPC-1, COLO 357, and T3M4 cells. In contrast, a similar pretreatment with rhTNF did not increase the long-term cytotoxic/cytostatic effects of rhIFN-gamma in any of the cell lines. These data suggest that, in some human pancreatic carcinoma cell lines, rhIFN-gamma may be especially useful in the long-term suppression of growth. Furthermore, brief pulses of rhIFN-gamma may also be especially efficacious when followed by a subsequent prolonged exposure of cells to rhTNF.

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

Recombinant interferon-gamma stimulates the production of human tumor necrosis factor in vitro.

Human peripheral blood monocytes in culture secrete tumor necrosis factor (TNF), which can be detected with a sensitive enzyme-linked immunosorbent assay system. When recombinant human interferon-gamma (rIFN-gamma) is added to cultured cells, TNF production is increased. rIFN-gamma is also able to sustain the elevated production level of TNF over a 4-day period. Recombinant interferon-alpha (rIFN-alpha) was unable to stimulate increased TNF production. Unstimulated mononuclear phagocytes and rIFN-alpha-treated cells decreased secretion of TNF over a 4-day interval. The stimulatory effect of rIFN-gamma was dose dependent and required both new RNA and protein synthesis and was independent of endotoxin in the tissue culture medium.

Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts.

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1kappa) and 3C5 (IgG2akappa), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

Alpha-globulins suppress human leukocyte tumor necrosis factor secretion.

The secretion of tumor necrosis factor (TNF) by human peripheral blood mononuclear cells was suppressed by either whole human plasma alpha-globulins or purified alpha 1-acid-glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin in a concentration-dependent manner. alpha 1-Antitrypsin was found to be the most suppressive of the purified proteins tested and completely blocked TNF release at concentrations above 1.25 mg/ml. Both alpha 1-acid glycoprotein and alpha 1-antitrypsin blocked TNF secretion by leukocytes which were simultaneously stimulated with either recombinant human interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). IFN-gamma- and LPS-activated cells were also susceptible to suppression mediated by these two alpha-globulins and the inhibition produced by 5 mg/ml alpha 1-antitrypsin was greater than that caused by either 1 microM prostaglandin E2 or 10 ng/ml transforming growth factor-beta 1. The level of TNF mRNA in TNF-secreting and alpha-globulin-suppressed cells was examined and found to be equal in both groups. The suppressive effect of whole alpha-globulins was confined to the inhibition of TNF secretion and these plasma proteins had no effect on the cytolytic activity of the recombinant cytokine as measured on murine L-929 target cells. Thus the alpha-globulins, which are a major fraction of the circulating plasma proteins, may function in TNF homeostasis by controlling TNF secretion without inhibiting the biological activity of the released cytokine.

STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors.

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.