Takashi Tsuruo

Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors

The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-specific manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein.

Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues. ATPase cytochemistry showed that these reactive skeletal muscle fibers were of the type I (slow-twitch) class. Other additional sites of C219 reactivity in rat tissues were found in pancreatic acini, seminal vesicle, and testis. Electrophoretic gel immunoblotting showed two protein bands reactive with MAb C219. In liver, MAb C219 reacted with a approximately 170 KD band. In skeletal and cardiac muscle, MAb C219 reacted with a approximately 200 KD band which migrated in the same position as myosin. This band also reacted with an antibody to skeletal muscle myosin. This result suggests that C219 may crossreact with the heavy chain of muscle myosin in cardiac and skeletal muscle. Because MAb C219 reacts with proteins other than P170, it should be used with caution in studies of multidrug resistance.

Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion

Abstract The development of acquired resistance to antineoplastic drugs and the associated broad cross-resistance to other agents frequently limits the effectiveness of chemotherapy. Ling and coworkers have demonstrated that Chinese hamster ovary (CHO) cells develop the phenotype of pleiotropic drug resistance which is manifest by a decrease in drug accumulation in these cells and hence a decrease in cytotoxicity (1). The role of drug accumulation and membrane glycoproteins in the expression of primary resistance and cross-resistance in human tumors is an area of active investigation (2–4). We have developed a series of human ovarian cancer cell lines with acquired resistance to melphalan, cisplatin, or adriamycin (5). These cell lines exhibit sensitivity/resistance profiles characteristic of pleiotropic drug resistance. In addition, the melphalan and cisplatin resistant variants are also cross-resistant to irradiation (6). Both the primary resistance to melphalan and the cross-resistance to irradiation in these cell lines can be reversed by lowering glutathione (GSH) levels in the cells with buthionine sulfoximine (BSO) (6,7). In the present study, the role of GSH in the expression of sensitivity to agents other than melphalan was examined by BSO-mediated depletion of GSH. In addition, the patterns of both primary resistance and cross-resistance were compared following GSH depletion in these cell lines.

Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization.

In many human cancers, the cyclin-dependent kinase inhibitor p27Kip1 is expressed at low or undetectable levels. The decreased p27Kip1 expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G1 arrest, in part by up-regulating p27Kip1. However, the role of Akt-dependent phosphorylation in p27Kip1 regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27Kip1. Screening p27Kip1 phosphorylation sites identified the COOH-terminal Thr198 residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27Kip1 through Thr198 only when it was phosphorylated by Akt. Although Akt also phosphorylated p27Kip1 at Ser10 and Thr187, these two sites were not involved in the binding to 14-3-3 proteins. p27Kip1 phosphorylated at Thr198 exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27Kip1 and cytoplasmic localization.

Domain Mapping Studies Reveal That the M Domain of hsp90 Serves as a Molecular Scaffold to Regulate Akt-Dependent Phosphorylation of Endothelial Nitric Oxide Synthase and NO Release

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation

We have investigated the effect of monoclonal antibodies (MAbs) specific for aminopeptidase N/CD13 on the invasion of human metastatic tumor cells into reconstituted basement membrane (Matrigel). The invasion of human metastatic tumor cells (SN12M renal‐cell carcinoma, HT1080 fibrosarcoma and A375M melanoma) into Matrigel‐coated filters was inhibited by an anti‐CD 13 MAb, WM15, in a concentration‐dependent manner. However, this MAb did not have any effect on tumor‐cell adhesion and migration to the extracellular matrices, which may be involved in tumor‐cell invasion. MAb WM15 inhibited the degradation of type‐IV collagen by tumor cells in a concentration‐dependent manner. We also found that WM15 inhibited hydrolysing activities towards substrates of aminopeptidases in 3 different tumor cells. Since our previous study indicated that bestatin, an aminopeptidase inhibitor, was able to inhibit tumor‐cell invasion, as well as aminopeptidase activities of murine and human metastatic tumor cells, cell‐surface aminopeptidase N/CD13 may be partly involved in the activation mechanism for type‐IV collagenolysis to achieve tumor‐cell invasion, and anti‐CD13 MAb WM15 may inhibit tumor‐cell invasion through a mechanism involving its inhibitory action on the aminopeptidase N in tumor cells.

Involvement of 14‐3‐3 proteins in nuclear localization of telomerase

Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post‐translational regulation of telomerase activity. Here we identify the 14‐3‐3 signaling proteins as human TERT (hTERT)‐binding partners. A dominant‐negative 14‐3‐3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT‐3A, a mutant that could not bind 14‐3‐3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1‐mediated nuclear export, or disruption of a nuclear export signal (NES)‐like motif located just upstream of the 14‐3‐3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild‐type hTERT, hTERT‐3A increased its association with CRM1. 14‐3‐3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14‐3‐3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES‐like motif.

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.

Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization

Breast cancer resistance protein (BCRP) is a half‐molecule ABC transporter highly expressed in mitoxantrone‐resistant cells. In our study we established PA317 transfectants expressing Myc‐tagged BCRP (MycBCRP) or HA‐tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70‐kDa protein in SDS‐PAGE under reducing condition, but migrated as a 140‐kDa complex in the absence of reducing agents. The 140‐kDa BCRP complex was heat‐stable but dissociated into 70‐kDa BCRP with the addition of 2‐mercaptoethanol. The 140‐kDa BCRP complex was immunoprecipitated with anti‐Myc antibody from the lysates of PA317 cells double‐transfected with MycBCRP and HABCRP. The 140‐kDa complex reacted with anti‐HA and anti‐BCRP antibodies and after the addition of reducing agents, a 70‐kDa protein reacting with anti‐Myc, anti‐HA and anti‐BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant‐negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug‐resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP‐transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP‐transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant‐negative inhibition as a new strategy to circumvent drug resistance.

Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer

Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), known to inhibit cyclooxygenase (COX), reduce the risk of colorectal cancer. COX is a key enzyme in prostaglandin biosynthesis, and two isoforms of COX, COX-1 and COX-2, have been identified. Recently COX-2 has been reported to frequently overexpress in colorectal neoplasms and to play a role in colorectal tumorigenesis and tumour progression. In this study, using immunohistochemistry, we examined COX-2 expression in advanced human colorectal cancer and its correlation with clinicopathological features. COX-2 expression was observed mainly in the cytoplasm of cancer cells in all the specimens examined, but some stromal cells and endothelial cells were also stained. According to the grade of COX-2 expression of the cancer cells, patients were divided into high- and low-COX-2 expression groups. High-COX-2 expression significantly correlated with tumour recurrence, especially haematogenous metastasis. These results suggest that a selective COX-2 inhibitor can be a novel class of therapeutic agents not only for tumorigenesis but also for haematogenous metastasis of cololectal cancer. To our knowledge, this is the first report on the correlation between COX-2 overexpression and recurrence of colorectal cancer.