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The transcription factor c-Myc is important for the control of cell growth, cell cycle progression, neoplasia, and apoptotic cell death. Recently, c-Myc-binding proteins, which bind either to the N-terminal domain or the C-terminal domain of c-Myc, have been proposed as the key molecules to realize the mechanisms of these multiple c-Myc functions. We report in the present study on another protein, Tiam1, which is a specific guanine nucleotide exchange factor of Rac1 and which binds to c-Myc and modulates several of its biological functions. We were able to detect the direct binding and in vivo association between c-Myc and Tiam1. The necessary role in this interaction of the Myc box II of c-Myc was revealed in the cell extracts. The additional discovery of the intranuclear localization of Tiam1 in Rat1 cells and in neuronal cells of the mouse brain suggests this interaction may occur in the nucleus. Overexpression of Tiam1 repressed the luciferase activity of c-Myc and also inhibited the c-Myc apoptotic activity through this protein-protein interaction. Taken together, we concluded that Tiam1 is another c-Myc regulator, working in the nuclei to control c-Myc-related apoptosis.

Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.

Purpose: Although chemotherapeutic protocols that include chloroethylnitrosoureas (CENUs), such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), have been a mainstay of treatment for glioblastomas, the clinical outcomes have been unsatisfactory. More effective chemotherapeutic protocols for these tumors will require clear delineation of more cytocidal and cytostatic chemotherapeutic drugs. Methods and Results: In this study, we measured the cytocidal effects of ACNU, cisplatin, actinomycin D, and staurosporine, administered within their therapeutic dose ranges, in the treatment of glioblastoma cells. As assessed by WST-1 colorimetric assay, the number of viable cells decreased markedly in T98G cultures treated with actinomycin D or staurosporine, to less than 20% of the level in control cultures at 72 h, but did not decrease or even increased after 6 days of treatment with ACNU. After treatment with cisplatin for 5 days, cell viability decreased to 30% of control. As assessed by fluorescence microscopic examination of nuclear staining by Hoechst 33258 and by electron microscopy, the majority of dead cells treated with actinomycin D, staurosporine, or cisplatin had morphologic features of apoptosis. Caspase-3 activity increased more than 20-fold in cells treated with actinomycin D, staurosporine, or cisplatin but increased less than fivefold in ACNU-treated cells. In addition to caspase-3 activation, western blot analysis demonstrated cleavage of caspase-2 during the apoptotic process. These findings indicate that actinomycin D and staurosporine potently induce apoptosis, whereas ACNU exerts mainly a cytostatic rather than a cytocidal effect. Conclusion: Actinomycin D and staurosporine and their derivatives are potentially effective chemotherapeutic agents against glioblastoma cells at least in vitro.

The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is ≈ 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of ≈ 5 mg·L−1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and α-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 °C, which was significantly higher than the optimal temperature (37 °C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.

Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.

Minor leucine tRNA species, tRNA and tRNA , from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567−585]. In the present study, these two tRNALeu species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA was found to correspond to the tRNA gene reported as su°6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627−644]. The first letter of the anticodon was identified to be 2‘-O-methylcytidine (Cm). tRNA was identified as the minor leucine tRNA that has been sequenced previously (tRNA ) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220−2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA . Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N* was found to be a 2‘-O-meth...

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.

BACKGROUND Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy. The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance. Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs. METHODS We present a 45-year-old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy. In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor. RESULTS Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance. On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low. Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable. Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression. For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis. Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16. CONCLUSIONS The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy.

Summary Two rat osteosarcoma cell lines, YROS-1 and YROS-2, were established from two experimental osteosarcomas and induced by internal irradiation with radioactive phosphorus. Both cell lines formed a monolayer cell sheet in vitro with focal piling. The YROS-1 cells were re fractile and spindle or polygonal in shape, whereas the YROS-2 cells were flat, spread and polygonal in shape. Ultrastructurally, the YROS-1 cells had well-developed rough-surfaced endo plasmic reticulum with focal pericellular deposition of calcified matrix, whereas YROS-2 had abundant polysomes and intracytoplasmic filaments. Both cell lines grew stably with population doubling times o f 23 and 39 h, respectively. Flow cytometry revealed that YROS1 was rich in proliferating cells compared to YROS-2, with a higher colony-forming efficiency. YROS-1 showed high alkaline phosphatase activity, while YROS-2 possessed low activity. When subcutaneously transplanted into lumbodorsal area o f athymic nude mice, only YROS-1 formed tumors with frequent lung metastasis.

A cell line, YP-MEL, was established from an intracranial malignant melanoma occurring in a neurocutaneous melanosis (NCMsis) patient. The established cell line was successfully cultured in serum-free medium with a doubling time of 41 h. The cells were refractile and small in size, with occasional pigmented giant cells. Histochemical and immunohistochemical features were compatible with common malignant melanoma and its cell line. Chromosome analysis revealed many supernumerary chromosomes and marker chromosomes including double minutes (DMs). When transplanted into nude mice, YP-MEL formed tumors histologically consistent with the original tumor. Addition of sera to the medium caused cellular spreading and elongation of cytoplasmic processes with an increase of melanin contents and tyrosinase activity. Because there was no melanoma cell line derived from a NCMsis patient, YP-MEL might be a beneficial tool for study on NCMsis.

Forced expression of the s-myc gene suppressed the tumorigenicity of rat RT4-AC tumor cells in nude mice, as reported previously. Polymerase chain reaction (PCR) analysis indicated that RT4-AC cells established from a tumor of the peripheral nervous system contain an activated neu gene with a T----A transversion in the transmembrane domain. Synthesis of a protein of 60 kd kd in RT4-AC cells was specifically inhibited by expression of the s-myc gene. These results strongly suggest that s-Myc suppresses the transforming activity of rat neural cells transformed by expression of the activated neu gene, and plays an important role in regulating expression of a cellular gene contributing to cell transformation, such as the gene that encodes the p60 protein.

The 14-3-3 family proteins are key regulators of various signal transduction pathways including malignant trans- formation. Previously, we found that the expression of the 14-3-3b gene is deregulated as well as c-myc gene in afla- toxin B1 (AFB1)-induced rat hepatoma K1 and K2 cells. To elucidate the implication of 14-3-3b in tumor cell growth, in this paper we analyzed the effect of forced expression of antisense 14-3-3b RNA on the growth and tumorigenicity of K2 cells. K2 cells transfected with antisense 14-3-3b cDNA expression vector diminished their growth ability in monolayer culture and in semi-solid medium. Expres- sion level of vascular endothelial growth factor mRNA was also reduced in these transfectants. Tumors that formed by the transfectants in nude mice were much smaller and histologically more benign tumors, because of their decreased level of mitosis compared with those of the par- ental cells. Frequency of apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was increased in the transfectant-derived tumors accompanying the inhibition of angiogenesis. In addition, over-expression of 14-3-3b mRNA was observed in various murine tumor cell lines. These results suggest that 14-3-3b gene plays a pivotal role in abnormal growth of tumor cells in vitro and in vivo. (3-6), Bcr/Bcr-Abl (7), polyoma middle T antigen (8), phos- phatidylinositol 3-kinase (9), protein kinase C (10), ASK-1 (11), BAD (12), Cdc25 (13,14), FKHRL1 transcription factor (15), keratin cytoskelton (16) and TERT telomerase subunit (17). However, the functional analysis of the 14-3-3 proteins in oncogenic transformation in vitro and in vivo is very limited. Recently Takihara et al. (18) reported that the enforced expression of 14-3-3b RNA in NIH3T3 cells confers on them a tumorigenicity in nude mice through the stimulation of mitogen-activated protein kinase (MAPK) cascade. Nonethe- less, there are conflicting reports that 14-3-3s related proteins are strongly down-regulated in SV40-transformed keratino- cytes, and in SV40- and v-Ha-ras-transformed epithelial cells (19,20). Moreover, the enforced expression of 14-3-3z in combination with ras or raf-1 cannot transform normal mouse fibroblasts (21). Thus, the real function of 14-3-3 pro- teins in oncogenic transformation is still vague. In the course of the study of aflatoxin B1 (AFB1) hepatocar- cinogenesis, we found that the expression of the 14-3-3b gene was deregulated together with the c-myc gene, which is known to cooperate with Raf-1 kinase for a cellular transformation, in AFB1-induced rat hepatocellular carcinoma K1 and K2 cells (22,23). Mutation of 14-3-3b gene locus was also detected in these cells (24). On the other hand, the mutation and/or deregu- lated expression of ras family oncogenes and suppressive oncogene such as p53 and Rb were not detected in K1 and K2 cells (24,25). Therefore, it is highly possible that 14-3-3b gene cooperates with c-myc gene in cellular transformation. In this paper, to investigate whether the over-expression of the 14-3-3b gene implicates in neoplastic phenotype of K2 cells, we established K2 cells expressing reduced level of 14-3-3b mRNA by the stable transfection with antisense 14-3-3b cDNA expression vector and analyzed their growth ability in vitro and in vivo. As we would expect, the antisense transfectants diminished their growth ability in the soft agar medium as well as in the monolayer culture. Tumors that formed by these transfectants in nude mice were much smaller and histologically more benign tumors. In these tumors, high frequency of apoptosis and inhibition of angiogenesis were also observed. Moreover, by the simultaneous addition of antisense 14-3-3b and c-myc oligodeoxynucleotides (ODNs) the K2 cell growth was diminished in a synergistic manner. We also found that the 14-3-3b gene was over-expressed in various tumor cell lines. Thus, it seems likely that the 14-3-3b gene plays an important role in tumor cell growth perhaps through the cooperation with the c-myc gene.

Background: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. Methods: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. Results: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P = .006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. Conclusions: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.

Background: In addition to traditional modalities such as surgical intervention and radiother­ apy, chemotherapy is a common therapeutic method for human malignant brain tumors. How­ ever, the effectiveness of chemotherapy is frequently hampered by cancer cell chemoresistance, resulting in an unsatisfactory outcome. To overcome this disadvantage, the proper selection of efficacious anticancer agents is required. Methods: The expression levels of chemoresistance-related genes, MGMT, mdr1, MRP, MTIIA and GST-n, in 28 surgical specimens of human brain tumors and in 10 human glioma cell lines were examined by Northern blot analysis. In addition, the SD1Q values of human gli­ oma cell lines against ACNU, COOP, ADM and VP16 were estimated by a cell survival assay. Results: The expression levels of each of the chemoresistance-related genes, except MRP, were generally higher in brain tumors than those in non-neoplastic brain tissues. MGMT expression correlated exclusively with ACNU resistance in all glioma cell lines examined (p = 0.0002). The transcriptional level of mdr1 in the tumor cells correlated with the 8010 values of VCR (p = 0.04) and ADM (p = 0.034). In contrast, the expression levels of MTIIA and GST-n did not correlate with resistance to any of the drugs tested. A correlation of MRP mRNA expres­ sion with multidrug resistance was not apparent in the 10 cell lines tested. Conclusions: The data indicate that knowledge of the expression levels of MGMTand mdr1 may be particularly useful for a more rational selection of drugs which are not influenced by these resistance genes and which have improved efficacy against human brain tumors.

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.

Proteolytic trimming of isoprenylated proteins, including Ras, at the C-terminal CAAX motifs is a key event in their activation. However, the protease responsible for the proteolysis has not been well characterized yet. In this study, we established a novel assay method for the enzyme using a fluorescent substrate, dansyl(Dns)-KSKTKC(S-farnesyl)VIM, with which we can assess the proteolytic activity with high sensitivity and more easily than by the former assay methods using radio-labeled substrates. Using this assay method, we purified the protease 104-fold from bovine brain microsomal membranes by Sepharose CL-6B gel filtration and DE-52 chromatography. The partially purified enzyme was shown to be an endoprotease specific to the farnesylated peptide and to have a K(m) value of 1.0 microM for Dns-KSKTKC(S-farnesyl)VIM. o-Phenanthroline and zinc chloride strongly inhibited the activity. Interestingly, however, m- and p-phenanthrolines were as effective as o-phenanthroline, indicating that the inhibition by o-phenanthroline is not simply due to its chelating action. The molecular mass of the protease was deduced to be 480 kDa by gel filtration. The enzymatic activity was lost during further attempts at chromatographic purification, but was partially recovered by mixing the chromatographic fractions which had apparently lost the activity. These results suggest that this protease consists of multiple subunits.

Objective A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. Methods We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. Results C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. Conclusion ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.

Changes in gene expression including that of c-fos occur following cerebral ischemia. Proto-oncogenes c-myc and s-myc and oncosuppressor gene p53 are known to induce apoptosis in some types of cells, whereas proto-oncogene bcl-2 inhibits apoptosis. Possible induction of mRNAs for c-myc, N-myc, s-myc, c-fos, p53 and bcl-2 was examined following focal ischemia in the rat anterior cortex, hippocampus, thalamus and cerebellum by Northern blot analysis. Animals were decapitated 1, 2, 6, 12, and 24 hours following the left middle cerebral artery (MCA) occlusion. In sham-operated control rats, the mRNAs for c-myc, N-myc, c-fos and p53 were present in the anterior cortex, hippocampus, thalamus on both sides, and in the cerebellum, whereas those for s-myc and bcl-2 were not. The c-myc gene expression was rapidly and markedly induced by the MCA occlusion in the ipsilateral anterior cortex, hippocampus and thalamus in a time-dependent manner. In these regions, the c-fos gene expression was also induced as early as 1 hour after the MCA occlusion. The p-53 mRNA was induced in the ipsilateral hippocampus at 24 hours after MCA occlusion. In contrast, mRNAs for N-myc, s-myc and bcl-2 were not induced following MCA occlusion. These results indicate a possibility that high-level expression of the c-myc gene may be involved in the ischemic cellular events including apoptosis.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the EGF family that binds to and stimulates phosphorylation of the EGF receptor (EGFR). In this study, we examined the cellular localization of HB-EGF gene transcripts and protein in adult rat forebrain. In situ hybridization studies showed that neurons in various regions, including cortex, hippocampus, and deep structures, express HB-EGF mRNA. Positively labeled cells were also present in white matter, which suggests that both neurons and glia express HB-EGF mRNA. Immunohistochemical studies with an antibody specific to proHB-EGF, a transmembrane form of HB-EGF, demonstrated ubiquitous immunoreactivity in neurons and glial cells in white matter. In view of the wide expression of its cognitive receptor, EGFR, in central nervous system neurons, our results suggest that HB-EGF is an endogenous ligand for EGFR in the central nervous system and may play an important role in physiological conditions.

Schwannoma-derived growth factor (SDGF) is a member of the epidermal growth factor (EGF) family, having mitogenic activity on rat astrocytes, fibroblasts and Schwann cells. The SDGF gene is significantly expressed in the newborn rat lung and in the adult rat sciatic nerve. However, except for one rat schwannoma cell line, from which SDGF and its cDNA were isolated, nothing is known about SDGF expression in established tumor cell lines. We examined the expression level of the SDGF gene in a variety of rat tumor cell lines by Northern blotting and found that it was increased in 11 of 25 established lines. The most abundant SDGF mRNA, which was about 50-fold higher than in the newborn rat lung, was expressed in rat liver adenoma dRLa74 cells. In rat glioma cell lines, such as C6, 9L and T9, and in the rat hepatoma dRLh84 and H411E cells, the SDGF expression level was about 10-fold higher than in the newborn rat lung. In 8 of 13 cell lines expressing SDGF mRNA, the EGF receptor (EGFR) gene, the product of which is regarded as a functional receptor of SDGF, was co-expressed. In addition, transfected gene-dependent anti-sense SDGF RNA expression under the control of the human metallothionein promoter significantly suppressed the in vitro growth as well as in vivo tumorigenicity of 9L glioma cells. Our results suggest that SDGF acts as an autocrine growth factor in the development and growth of rat tumors such as gliomas.

Mammalian cells contain an intron-less myc gene, such as the rat s-myc gene and human myc L2 gene, which are expressed in rat embryo chondrocytes and human testis, respectively. Our recent findings demonstrated that s-Myc expression suppresses the growth activity and tumorigenicity of glioma cells, indicating that s-Myc acts as a negative regulator in tumor growth. In addition, we found that s-Myc overexpression can effectively induce apoptotic cell death in human and rat glioma cells without serum deprivation, which is distinct from c-Myc-mediated apoptosis.

The s-myc gene is a unique myc family gene without introns that was isolated from a rat genomic library using the –-myc exon 3 region as a probe. Significant expression of the s-myc gene was detected in rat embryo chondrocytes committed to programmed cell death. Gene transfection experiments showed that s-myc expression in rat and human glioma cells killed the cells by apoptosis induction. These obser–a-tions, indicating that s-Myc can act as an apoptosis inducer in –i–o and in –itro, suggest that the s-myc gene might be useful for gene therapy of gliomas. To test this possibility, we introduced the s-myc gene (pCEP4smyc), linked to the cytomegalo–i-rus promoter, into glioma-bearing rats. Surprisingly, injection of pCEP4smyc plasmid DNA together with rat 9L or C6 glioma cells completely pre–ented 9L- or C6-deri–ed tumor formation in the brain or the hind leg of rats. A similar dramatic antitumor effect induced by s-myc gene expression was obser–ed at a site distant from the original site at which the s-myc gene and glioma cells were coinjected. These results indicated that s-myc expression has the potential to elicit a host antitiumor immune response.

✓ The authors investigated the roles of endothelin (ET)-1 and the ETA receptor in the pathogenesis of delayed cerebral vasospasm following subarachnoid hemorrhage (SAH). A study was made of the preventive effect of a novel ETA receptor antagonist, BQ-123, on vasospasm and the expression of the ETA receptor messenger ribonucleic acid (mRNA) using a canine two-hemorrhage SAH model. Continuous intrathecal administration of BQ-123 (5 × 10−6 mol/day) prevented narrowing of the basilar artery on Day 7 after SAH in 97.6% of cases in the study group versus 70.7% of cases in the control group (p < 0.05). While expression of the mRNA-coding ETA receptor was not detected in the control animals, it markedly increased on Day 3 after SAH and was also detected on Day 7. The results suggest that endothelin-1 and the ETA receptor participate in the pathogenesis of delayed cerebral vasospasm following SAH.

The dominant +1 frameshift suppressors sufA6, sufB1 and sufB2, in Salmonella typhimurium act at runs of C and affect tRNA(Pro)1, tRNA(Pro)2 and tRNA(Pro)2, respectively. A recessive +1 frameshift suppressor, sufC, has a similar suppressor specificity (Riddle, D.L., and Roth, J.R., Mol. Biol. 66, 483 and 495, 1972). We show that sufC strains harbour two frameshift suppressors of which one, sufX201, is allelic to sufB. We cloned the sufB+ wild type allele and by recombination in vivo the mutations sufB1, sufB2 and sufX201. Determination of the DNA sequence revealed that the sufB1 and sufB2 mutations result in an extra G in the anticodon loop of the minor tRNA(Pro)2. The sufX201 mutation results in a base substitution (G43 to A43) in the anticodon stem of this tRNA. Although the sufB1 and sufB2 mutations were earlier shown to be dominant, the sufB+ wild type allele on multi copy plasmid inhibited the chromosomal sufB1, sufB2 and sufX201 mediated frameshift suppression but not that mediated by the dominant sufA6 mutation. These results are discussed in view of the possible coding specificity of these mutated tRNAs. The DNA sequence showed a potential consensus promoter sequence upstream of the structural gene for tRNA(Pro)2 and downstream a dyad symmetrical structure followed by a T cluster, a possible rho-independent termination signal. The Salmonella tRNA(Pro)2 gene is identical to the Escherichia coli counterpart reported by Komine, Y. et al. (J. Mol. Biol. 212, 579-598, 1990). While the 5' flanking sequence similarity between the two species is about 83%, the similarity of the 3' flanking sequence is only 42%. Still, the Salmonella tRNA(Pro)2 gene has a rho-independent transcriptional termination signal similar to the one present in E. coli tRNA(Pro)2 gene.

Northern blot analysis with O6-methylguanine-DNA methyltransferase (MGMT) cDNA as a probe was used to analyze the MGMT activity regulating drug resistance of human cells to chloroethylnitrosoureas (CENUs). By this method, the expression levels of MGMT mRNA in six human glioma cell lines and 12 human brain tumor tissues from surgical specimens were determined. These MGMT mRNA levels were compared with the SD10 values of the tumor cells, estimated by cell survival assay, which indicated their resistance to the anticancer drug, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). Human brain tumors that were highly resistant to ACNU, such as glioblastoma Gbl1 and metastatic brain tumor Col1 with SD10 values (microM) of above 100, expressed markedly increased amounts of 0.95 kb MGMT mRNA. In contrast, tumor cells such as U-87MG, U-251MG, U-343MG, U-373MG and SF-126 with SD10 values of under 14 indicating low resistance to ACNU scarcely synthesized any MGMT mRNA. These results indicated that the level of expression of MGMT mRNA in human brain tumors determined by Northern blot analysis truly reflects their cellular resistance to ACNU. Thus the Northern method with MGMT cDNA probe reported here is a practical and reliable method for estimation of cellular resistance to CENUs such as ACNU and for screening the chemotherapeutic response to CENUs of human brain tumors.

Among Moloney murine leukemia viruses (Mo-MuLVs) having stop codons other than UAG at the gag-pol junction, Mo-MuLV with UAA, but not with UGA, had a replication disadvantage. Mo-MuLV with a glutamine codon (CAG) at the junction did not replicate. A revertant of this virus consisted of the original virus and a virus with a deletion of the pol region. Protease and Pr65gag encoded by their respective genomes complemented each other.

A novel enzyme which catalyzes the transfer of the 3-amino-3-carboxypropyl group into tRNA to form 3-(3-amino-3-carboxypropyl)uridine was isolated from Escherichia coli . The enzyme required S-adenosylmethionine as donor molecule.

Two species of glutamine tRNA were isolated from mouse liver and their nucleotide sequences were determined. The minor glutamine tRNA(tRNA(UmUGGln)) that possesses UmUG (where Um stands for 2'-O-methyluridine) as the anticodon sequence was found to have suppressor activity for the UAG termination codon of tobacco mosaic virus RNA in a rabbit reticulocyte in vitro translation system. The amount of this suppressor glutamine tRNA in mouse liver was 1-2% of the amount of the major glutamine tRNA(tRNA(CUGGln)) that has the CUG anticodon sequence, but it was markedly increased in NIH 3T3 cells infected with Moloney murine leukemia virus and in Ehrlich ascites cells. These results support the hypothesis that tRNA(UmUGGln) actually functions in vivo as a suppressor tRNA that recognizes the UAG termination codon located at the gag-pol gene junction of Moloney murine leukemia virus and results in the synthesis of the virus-encoded protease.

Avarol is a sesquiterpenoid hydroquinone, which displays no inhibitory potencies on mammalian DNA polymerases alpha, beta, and gamma, on mammalian RNA polymerases I, II, and III, or on reverse transcriptases from Moloney murine leukemia virus (Mo-MuLV) and from HIV. For a further elucidation of the antiviral effect of Avarol, we used NIH-3T3 cells infected with Mo-MuLV as a model system. The results show that in uninfected NIH-3T3 cells Avarol (i) causes a 50% reduction of the growth rate only at the high concentration of 29.6 microM and (ii) is accumulated in the cytoplasm close to the nucleus. At the much lower concentrations of 1-3 microM, Avarol causes an almost complete inhibition of viral progeny release. Moreover, it is shown that at 3 microM Avarol, the increase of the Mo-MuLV-induced UAG suppressor glutamine tRNA (tRNA(UmUGGln) was reduced to the normal level. Dot blot hybridization studies revealed that Avarol displays no inhibitory activity on cellular and viral mRNA synthesis. Taking the processing pathway of viral polyprotein Pr180gag,pol to p80 (reverse transcriptase) as an example, our Western blotting experiments showed that the final maturation process, conversion of p110 to p80, is inhibited in Avarol-treated cells. From these data we conclude that Avarol prevents the suppression of the UAG termination codon at the gag-pol junction of the retroviral genome. The functional consequence of this event is very likely an inhibition of the readthrough of the retroviral protease gene which overlaps the pol and gag genes, resulting in the reduction of the protease synthesis which is necessary for the viral proliferation.

The primary structure of Escherichia coli tRNA UUR Le which recognizes the UU series of codons has been determined. The sequence is pG-C-C-C-G-G-A-s4U-G-G-U-G-G-A-A-D-C-Gm-C-D-A-G-A-C-A-C-A-A-G-G-G-A-psi-U-N-A-A-ms2i6A-A-psi-C-C-C-C-U-C-G-G-C-G-G-C-G-U-U-C-G-C-G-C-U-G-U-G-C-G-G-G-T-psi-C-A-A-G-U-C-C-C-G-C-U-C-C--G-G-G-U-A-C-C-A. The chain length of tRNA UUR Leu is 87 residues, the same as other E. coli tRNA Leu s and T4 phage-coded tRNA Leus. Its sequence is especially similar to that of E. coli tRNA2 Leu in the Darm and T psi C arm regions. E. coli tRNA UUR Leu contains an unknown modified nucleoside in the first position of the anticodon and was shown by mass spectrometry and chemical degradation to be an adenosine derivativee. Addition of tRNA UUR Leu to a cell-free protein-synthesizing system with high Mg2+ concentration resulted in the formation of polyleucine miscoded by poly(U), indicating that the unknown modified nucleoside exhibits a tendency to recognize U under certain conditions.

A 5 kbp DNA fragment containing the tRNAPro1 gene from Escherichia coli was cloned into Charon 21A phage and sequenced by the M13 DNA sequencing technique. When the cloned DNA fragment was used as a template for in vitro transcription with E. coli RNA polymerase, a tRNAPro1 precursor of 120 nucleotide residues was obtained. The tRNAPro1 gene transcribed as a single transcription unit was followed by two unusual repeating sequences, both of 108 bp. These two repeating sequences were separated by a 60 bp spacer sequence. The 5'-portion of each repeating sequence overlapped 19 bp of the 3'-terminal region of the tRNAPro1 gene just like the repeating sequences in the E. coli tRNATyr1 gene. A rho-independent termination was present in the first repeating unit.

A cloned 2.1 kb fragment of rat DNA hybridized to purified tRNAAsp has been sequenced. The result revealed that in addition to the putative gene for tRNAAspGAU(C), the fragment contained the tRNAGlyGGA(G) and tRNAGluGAG genes. The genes for tRNAAsp, tRNAGly and tRNAGlu have the same polarity, are arranged in this order and are regularly separated by DNA regions of about 450 bp. These rat genes contain neither intervening sequences nor the CCA sequence expected in the 3'-end of the mature tRNA. As observed in lower eukaryotic tRNA genes, the 5'-flanking regions of the three rat genes do not have any significant sequence homology as a regulatory element. In the 3'-flanking region, the sequences CTTTTTG and CTTTTG are present 11 bp downstream from the 3'-end of the genes for tRNAAsp and tRNAGly, respectively. The same CTTTTG sequence is repeated twice in regions 47 and 60 bp away from the tRNAGlu gene. The short T cluster common to the three genes might be the transcription termination site as in lower eukaryotic tRNA genes.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

Nucleotide sequences of normal mouse liver tRNAPhe and tumor-specific tRNAPhes isolated from Ehrlich ascites tumor and neuroblastoma cells were examined by post-labeling techniques. The results showed that their sequences are identical, except for changes in post-transcriptional modifications that are located in the anticodon region. Normal mouse liver tRNAPhe contained Cm32, Gm34 and YOH37. On the other hand, tumor-specific tRNAPhes were found in one of two possible configurations: 1) Cm32, Gm34 and Y*OH37 (under-modified YOH) or 2) C32, G34 and m1G37. The ratio of the two forms of tRNAPhes differed in different tumor cells; Ehrlich ascites tumor tRNAPhe had mainly Y*OH-containing tRNAPhe whereas neuroblastoma tRNAPhe has predominantly m1G-containing tRNAPhe. It was concluded that tumor-specific tRNAPhes are products of different extents of modification, rather than of new tRNA transcription.

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post-labeling techniques. Two of these tRNAsGln show unusual codon recognition: a previously isolated tRNAGlnUmUA and a second species with CUA in the anticodon (tRNAGlnCUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNAGlnUmUA reads the UAA codon of α-globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNAGlnCUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAsGln is their unusually strong readthrough efficiency, e.g. purified tRNAGlnCUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNAGln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNAGlnUmUG and tRNAGlnUmUA is 81%, whereas that between tRNAGlnCUA and tRNAGlnUmUA is 95%, indicating that the two unusual tRNAsGln evolved from the normal tRNAGln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.

The nucleotide sequence of an E. coli isoleucine tRNA (tRNAIle minor) specific for the codon AUA was determined by postlabeling procedures using only 2.5 micrograms (0.05 A260 unit) of the material. The sequence was pG-G-C-C-C-C-U-s4U-A-G-C-U-C-A-G-U-Gm-G-D-D-A-G-A-G-C-A-A-G-C-G-A-C-U-N+-A-U-t6A-A-psi-C-G-C-U-U-G-m7G-acp3U-C-G-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-C-A-G-G-G-G-C-C-A-C-C-AOH. The nucleotide sequences in the regions of the D arm and T psi C arm of tRNAIle minor were quite similar to the corresponding regions of tRNAIle major. However, the sequences in the CCA stem and anticodon stem of tRNAIle minor were different from those of tRNAIle major. The overall homology between the two isoleucine tRNAs was 68%. E. coli tRNALys, tRNAMet, tRNAValIIA and tRNAArg also have relatively high sequence homology with tRNAIle minor.

The nucleotide sequences of three proline tRNAs from Salmonella typhimurium were determined by post-labeling procedures. The three proline tRNAs had almost identical sequences in the D-arm and T psi C-arm, and all contained 1-methylguanosine next to the 3'-end of the anticodon. The anticodon sequences of tRNAPro1, tRNAPro2 and tRNAPro3 were 5'-CGG-3', 5'-GGG-3', and 5'-VGG-3', respectively. The nucleotide sequence homologies of tRNAPro2 to tRNAPro1 and tRNAPro3 were 68% and 78%, respectively.