Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells after treatment with tumor promoters, chemical carcinogens, metal salts, and heat shock.

The synthesis of a Mr 32,000 protein (p32) is enhanced as early as 2 h after the addition of tumor-promoting phorbol esters to BALB/c 3T3 cells. Among various compounds tested thus far, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, L-ascorbic acid, sodium deoxycholate, p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, 3,3'-diaminobenzidine, and some of the metal salts stimulated the synthesis of p32 to varying extents. p32 might be one of the heat shock proteins because its synthesis was also stimulated by heat shock or sodium arsenite. The synergisms of the effects of different compounds on p32 synthesis suggest that the expression of a p32 gene is regulated through at least three pathways. Possible roles of protein kinases in p32 gene expression are discussed.

Defects of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in initiated clones derived from BALB/c 3T3 mouse fibroblasts.

Two-stage carcinogenesis is involved in the transformation of mouse fibroblasts BALB/c 3T3 cells. In order to investigate the role of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase at the stage of initiation, the following experiments were carried out: (a) two initiated clones (M14, M20) which exhibit 12-O-tetradecanoylphorbol-13-acetate-dependent growth in soft agar medium were isolated from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. The activity of cAMP-dependent protein kinase in M14 was reduced while that in M20 was similar to the level in parental cells. However, cAMP-binding activity to a regulatory subunit of cAMP-resistant clones were isolated from 4-nitroquinoline oxide- or ethyl methanesulfonate-treated cells. These clones have reduced activities in both cAMP-binding and cAMP-dependent protein kinase itself. Two of three cAMP-resistant clones were proved to be able to grow in soft agar medium only in the presence of 12-O-tetradecanoylphorbol-13-acetate.

Isolation and characterization of a complementary DNA clone for a Mr 32,000 protein which is induced with tumor promoters in BALB/c 3T3 cells.

The synthesis of a protein of Mr 32,000 (p32) is enhanced by various tumor promoters, chemical carcinogens, metal salts, and heat shock in BALB/c 3T3 cells. We have isolated a complementary DNA (cDNA) clone for p32 from a lambda gt10 library of BALB/c 3T3 cells. The library was constructed from mRNA extracted from the cells treated with sodium arsenite, which stimulates the p32 expression most effectively among various agents so far tested. Having screened this library differentially with probes which represent induced and uninduced mRNA populations for p32, we first obtained a partial p32 cDNA clone and have subsequently succeeded in the isolation of a cDNA clone containing the entire coding sequence. RNA blot analysis has shown that p32 mRNA is induced as early as 0.5 h after the addition of 12-O-tetradecanoyl-phorbol-13-acetate or sodium arsenite. Computer-assisted comparison with GenBank data has revealed a striking similarity in the nucleotide sequences between cDNAs of p32 and rat heme oxygenase. These results strongly suggest that p32 is a mouse homolog of this enzyme.

Increase in the synthesis of a Mr 32,000 protein in BALB/c 3T3 cells treated with tumor-promoting indole alkaloids or polyacetates.

The synthesis of a unique protein with a molecular weight of 32,000 (p32) in BALB/c 3T3 cells has been shown previously to increase after treatment with potent tumor-promoting phorbol esters (Hiwasa et al., Proc. Natl. Acad. Sci. U. S. A., 79: 1800, 1982). In the present study, two new classes of tumor promoters which are structurally different from phorbol esters were investigated for their potencies to enhance p32 synthesis. Teleocidin, dihydroteleocidin B, and lyngbyatoxin A, which are indole alkaloid tumor promoters, enhanced p32 synthesis to the same extent that 12-O-tetradecanoylphorbol-13-acetate did. However, no increase was observed by treatment with the biologically inactive hydrolysate of teleocidin. Polyacetate tumor promoters such as aplysiatoxin and debromoaplysiatoxin also stimulated p32 synthesis, but their effective concentrations were higher than those of 12-O-tetradecanoylphorbol-13-acetate. When 3T3 cells were treated with a combination of two of the three tumor promoters, TPA, teleocidin, and aplysiatoxin, no synergistic effect of p32 synthesis was observed. This implies that these tumor promoters enhance the synthesis of p32 through the same mechanism.

High levels of expression and nuclear localization of interleukin-1 beta converting enzyme (ICE) and CPP32 in favorable human neuroblastomas.

Neuroblastomas frequently show spontaneous regression and differentiation, which may at least partly be regulated by signaling through nerve growth factor and its receptors, TRK-A and p75LNTR. We studied 52 neuroblastic tumors to test whether the cell death-related proteases, interleukin-1 beta converting enzyme (ICE), CPP32, and Ich-1, were involved in the regression of the tumors. High levels of expression of ICE and CPP32 were significantly correlated with a high level of TRK-A expression, single copy of N-myc, younger age, lower stages, and better prognosis. The immunohistochemical studies and Western analyses as well as the terminal dUTP-biotin nick end labeling (TUNEL) method revealed that both ICE and CPP32 were translocated from the cytoplasm into the nuclei in regressing, apoptotic tumor cells. Our results suggest that ICE and CPP32 cysteine proteases may play an important role in regulating the apoptotic process of the favorable neuroblastomas.

A partial cDNA for a novel protein which has a typical E-F hand structure.

We cloned a partial cDNA which includes an open reading frame of 459 bp long from a mouse fibroblast cDNA library. The deduced amino-acid sequence showed a partial homology with several calcium-binding proteins. The clone possibly encodes a novel calcium-binding protein whose domain adopts the E-F hand structure.

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells.

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.

Loss of Raf-1-binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151.

In order to elucidate the molecular events in signal transduction, examination of the interaction between Ras and Raf-1 seems crucial. Many Raf-1 mutants have been investigated in terms of their binding activities to Ras, where only a few Ras mutants have been examined thus far. We have investigated the Raf-1-binding activities of v-Ha-Ras and 21 insertion/deletion mutants of this protein. The results show that the mutants have varying levels of Raf-1-binding activity that are related neither to their transforming activity nor to their guanine nucleotide-binding activity. Deletion in the effector domain of Ras did not completely abolish Raf-1-binding, whereas the deletion in amino acid residues 64-72 or 143-151 resulted in complete loss of Raf-1-binding activity.

v-Ha-Ras insertion/deletion mutants with reduced protease-inhibitory activity have no transforming activity.

We have purified 26 insertion/deletion mutants of v-Ha-ras oncogene products produced by Escherichia coli and investigated their protease-inhibitory activity toward papain and cathepsins B and L. Ki values for papain were relatively similar among the mutants, however, those for cathepsins B and L varied up to 10-fold. Among them, four mutants, 1-48 LIR 54-189, 1-110 LIS 112-189, 1-130 PDQ 146-189 and 1-155 LIR 166-189, showed significant reduction in the inhibitory activity toward cathepsin L and these four mutants have lost transforming activity toward NIH3T3 mouse fibroblasts. However, some other mutants also showed no transforming activity in spite of possession of the potent protease-inhibitory activity, suggesting that the protease-inhibitory activity of Ras might be necessary but not sufficient for its biological activity.

Enhancement of catalytic activities of serine proteases by tripeptides compounds.

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.

c-Ha-ras gene products are potent inhibitors of cathepsins B and L.

c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.

Suppression of okadaic acid-induced apoptosis by overexpression of calpastatin in human UV(r)-1 cells.

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UV(r)-1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5-fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector-transfected cells but not in calpastatin-transfected cells. This indicates that OA-induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA-induced hyperphosphorylation of c-Jun was inhibited in calpastatin-transfected cells. This might be involved in the resistance to OA-induced cell death in calpastatin-overproducing cells.

Down-regulation of protein kinase C alpha and gamma and enhanced TPA-induced neurite formation in DAN-transfected neuroblastoma cells.

DAN gene was first isolated by differential screening between rat 3Y1 and v-src-transformed 3Y1 cells and showed a tumor-suppressive activity toward v-src-transformed 3Y1 cells. When DAN-transfected neuroblastoma cells were treated with a tumor promoter phorbol ester, TPA, neurite-like processes appeared within 2 h whereas no apparent change was observed in the parent and vector-transfected cells up to 8 h. This suggests some difference in TPA-receptor, protein kinase C (PKC), between DAN-transfectants and the control cells. DAN-transfected SH-SY5Y cells showed complete loss in PKCalpha and a large decrease in PKCgamma. Similar down-regulation in PKCalpha and PKCgamma was also observed in DAN-transfected Ha-ras-transformed NIH 3T3 cells. The decreased level of PKCalpha was partially recovered after treatment with a calpain inhibitor, ZLLH. A 150-kDa proteolytic product of a calpain-specific substrate, non-erythroid alpha-spectrin, was detectable in DAN-transfected SH-SY5Y cells but not in the parent or vector-transfected control cells. This suggests that DAN-transfected cells contain activated calpain which may cause down-regulation of PKC and hence induce the altered TPA response.

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors.

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.

Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products.

Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.

Interleukin-1 beta converting enzyme (ICE) is preferentially expressed in neuroblastomas with favourable prognosis.

To determine whether interleukin-1 beta converting enzyme (ICE) plays a role in the programmed cell death of neuroblastoma, we studied ICE expression in primary tumours. In patients in stages I, II and IVS, ICE mRNA was detected in 22 of 32 (69%) tumours, while only 5 of 26 (19%) tumours expressed ICE in stages III and IV (P < 0.001). ICE mRNA was expressed in 27 of 47 (57%) tumours without MYCN amplification, but it was not detected in any tumours with MYCN amplification (P < 0.01). Immunohistochemically, the cytoplasm was stained in all 15 neuroblastomas examined. The nuclei were stained in 12 neuroblastomas without MYCN amplification, whereas only 1 of 3 tumours with MYCN amplification had positive staining in the nuclei. In ganglioneuromas, high levels of ICE mRNA were expressed, but immunostaining showed that the protease expression was confined to the cytoplasm. These observations suggest that ICE may be associated with the spontaneous regression often seen in favourable neuroblastomas and that localisation of ICE protease in the cell may be important for the cell death pathway. Double staining for ICE and TUNEL showed that they were co-localised in some nuclei, but the distribution of ICE protease expression was not necessarily the same as that of DNA fragmentation, suggesting that the protease expression probably preceded DNA fragmentation during the apoptotic process. ICE may play an important role in regulating the apoptotic process of neuroblastoma.

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase.

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

Nuclear localization of procathepsin L/MEP in ras-transformed mouse fibroblasts.

It has been well-documented that secretion of procathepsin L is enhanced in ras-oncogene-transformed cells. In the present study, intracellular localization of cathepsin L was investigated by cell fractionation using Nonidet P-40 followed by immunoblot analysis. The results showed that a significant amount of procathepsin L was detectable in the nuclear fraction of Ha-ras, Ki-ras- and erbB2-transformed NIH3T3 mouse fibroblasts while procathepsin L was detected only in the cytoplasmic fraction of NIH3T3 cells and v-mos-transformed cells. These results suggest that the processing and translocation of cathepsin L are seriously impeded in ras- and erbB2-transformed cells.

Potent growth-suppressive activity of a serine protease inhibitor, ONO-3403, toward malignant human neuroblastoma cell lines.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 and FO-349 are its derivatives. The effects of these compounds on the proliferation of 13 human neuroblastoma cell lines were investigated in vitro by MTT colorimetric assay. The half maximum inhibition concentrations of ONO-3403 varied between 22 and 90 microg/ml while those of FOY-305 and FO-349 were higher than 100 microg/ml. ONO-3403 showed higher growth-inhibitory activity for N-myc-amplified neuroblastomas as compared with that for non-amplified cells. Since N-myc amplification in neuroblastomas is well correlated with a poor prognosis, ONO-3403 could be an effective anticancer drug for malignant neuroblastomas.