Binding of pEL98 protein, an S100-related calcium-binding protein, to nonmuscle tropomyosin.

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was placed into a bacterial expression vector to produce a fusion protein containing glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathione-Sepharose beads bearing GST/TM2 were incubated with [35S]methionine-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The binding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning residues 39-107 on TM2 by using several COOH-terminal and NH2-terminal truncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography on a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, two-dimensional polyacrylamide gel analysis and a binding experiment revealed that the 10-kD protein was identical to a calcium-binding protein derived from mRNA named pEL98 or 18A2 that is homologous to S100 protein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed that some of the 10-kD protein was associated with the Triton-insoluble cytoskeletal residue in a Ca(2+)-dependent manner. Furthermore, immunofluorescent staining of NIH 3T3 cells showed that some of the 10-kD protein colocalized with nonmuscle TMs in microfilament bundles. These results suggest that some of the pEL98 protein interacts with microfilament-associated nonmuscle TMs in NIH 3T3 cells.

[Surgically treated pleomorphic carcinoma of the lung].

We experienced 3 resected cases of pleomorphic carcinoma of the lung. Each cases were 74-year-old man (case 1), 74-year-old woman (case 2) and 69-year-old man (case 3). Two patients (case 1 and 2) were histologically diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with giant cell carcinoma. One patient (case 3) was similarly diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with adenocarcinoma and squamous cell carcinoma. Although lymph nodes metastasis were not recognized in all patients, invasion to vessels were recognized in 2 patients (case 1 and 3). In one patient (case 1), recurrence was recognized at contralateral side 1 month after surgery and he died of other disease 2 months after surgery. The other 2 patients were alive without recurrence 24 and 5 months after surgery. Recently it is reported that recurrence is recognized at early phase after surgery and prognosis is poor in a case with vessel invasions in spite of pathological NO state. Since one patient (case 3) had nonmetastatic lymph nodes with vessel invasions, careful observation is considered to be necessary.

[Approach by clamshell incision for bilateral pulmonary metastasis].

In recent years thoracotomy by "clamshell incision" has frequently been chosen for heart-lung transplantation, bilateral lung tumors and mediastinal tumor merging into lung tumor because this approach provides very good visibility to access the whole bilateral lung including the lower lobe and mediastinal organs. In our hospital, 4 patients underwent bilateral thoracotomy by clamshell incision for pulmonary metastasectomy between 2001 and 2005. All cases had bilateral pulmonary metastases, and multiple wedge resection was performed. All lesions that were planned for resection on preoperative computed tomography (CT) could be resected. Regarding the surgical approach to bilateral pulmonary metastases that did not need lobectomy, clamshell incision is one of the useful approaches that can allow wedge resection anywhere in the whole lung. In cases that are expected multiple procedures, bilateral thoracotomy by clamshell incision is recommended because it allows another route for thoracotomy at reoperation.

[Clinical experience with 16 cases of mediastinal lymphoma].

Sixteen cases of mediastinal lymphoma in our hospital were reviewed clinically. There were 7 men and 9 women, whose mean age is 35 years old. The histological types were non-Hodgkins' disease in 12 (B-cell type in 6 and T-cell type in 6), Hodgkins' disease in 2, mucosa-associated lymphoid tissue (MALT) lymphoma in 2. All cases except 1 in which percutaneous needle biopsy was performed were diagnosed histologically. Even small specimens by percutaneous needle biopsy can be helpful in diagnosing histological type and subtype with immunohistlogy, recommending percutaneous needle biopsy as an mitial step for diagnosis. When histological diagnosis can not be made by needle biopsy, open biopsy should be done.

[Clinical analysis of tracheoplasty and bronchoplasty in primary lung cancer].

In our department, there were 482 thoracic surgeries for primary lung cancer between 1994 and 2007. We clinically reviewed cases that underwent tracheoplasty or bronchoplasty (n = 22, 4.6%). The patients consisted of 21 males and 1 female (66.5 +/- 12.0 years-old). All patients were smokers. The tissue forms were 19 squamous cell carcinomas, 2 adenocarcinomas, 1 large cell carcinoma, 1 adenoid cystic carcinoma and 1 carcinoid, including 2 multiple carcinomas. Sleeve resections involved the trachea in 1, upper lobes in 13, lower lobes in 3, upper-middle lobes in 2 and intermediate bronchus in 1. Wedge resections were performed in the upper lobes in 2. Fourteen reconstructions were performed. We ordinarily sutured the trachea and bronchus in any case, using a single outside knot. There was no leakage at the anastomosis. There were 2 hospital deaths. There were 4 cancer deaths, including 2 local recurrences. There were 4 patients demonstrating stenosis post operatively. There were 3 stenoses among 4 preoperative radiation therapies. We considered that radiation therapy disturbed the repair of the anastomosis. There were 8 pneumonia patients who developed post operatively. There were 2 operative hospital deaths among 3 angio-bronchoplasties without coverage. Recently, we have routinely covered the anastomosis at the reconstruction site and have not experienced any major complications.

Differential expression of a tropomyosin isoform in low- and high-metastatic Lewis lung carcinoma cells.

Two-dimensional electrophoretograms of newly synthesized polypeptides from low-metastatic (P29) and high-metastatic (D6) Lewis lung carcinoma cells were compared. The results showed that the synthesis of tropomyosin 2 (TM2) was significantly less in D6 cells than in P29 cells. Furthermore, suppression of TM2 synthesis was induced in P29 cells during incubation in medium containing dimethyl sulfoxide or butyric acid, which induced the metastatic phenotype of P29 cells. These results suggest that the suppression of TM2 synthesis is linked to the metastatic potential of Lewis lung carcinoma cells.

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.

Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells.

We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.

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.

Tumor-suppressive activity of N03 gene product in v-src-transformed rat 3Y1 fibroblasts.

The expression of the N03 gene was significantly reduced in a variety of transformed cells, compared with that in parental rat 3Y1 fibroblasts. To understand the possible role(s) of its product, we have obtained transfectants expressing N03 gene from v-src-transformed 3Y1 cells. Two clones, which overexpressed N03 mRNA, showed a flatter phenotype, a reduced rate of growth, and a decreased ability to grow in soft agar. One of the clones which maintained N03 expression was completely suppressed in forming tumors in nude mice. These data suggest that the N03 gene product may have a suppressive ability on transformed phenotypes.

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.

Characterization of messenger RNA for fructose 1,6-bisphosphate aldolase A isozyme of rat ascites hepatoma AH 7974 cells.

The messenger activity for fructose 1,6-bisphosphate aldolase (EC4.1.2.13) (aldolase) A isozyme has been characterized in the polysome- or the messenger RNA-directed, protein-synthesizing system using the pH 5 fraction of rat liver or wheat germ extracts, respectively. The subunit of aldolase A synthesized in vitro was detected by immunoprecipitation with anti-aldolase A antibody raised in chickens followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The synthesis of the enzyme depended on the addition of polysomes or polyadenylate-containing RNA of rat ascites hepatoma AH 7974 cells which show a complete shift of aldolase isozyme to type A, whereas polysomes of adult rat liver were inactive. The messenger activity for aldolase A was present exclusively on free polysomes but absent on membrane-bound polysomes and in the soluble supernatant fraction of AH 7974 cells. The size of aldolase A messenger RNA determined by formamide-containing sucrose density gradient centrifugation was approximately 5.8 X 10(5) daltons corresponding to 1650 nucleotides. Taking into account the number of amino acid residues in the aldolase A subunit, approximately 400 nucleotides correspond to the noncoding region of aldolase A messenger RNA.

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.

Therapeutic efficacy of the suicide gene driven by the promoter of vascular endothelial growth factor gene against hypoxic tumor cells.

We examined whether herpes simplex virus thymidine kinase (HSV-TK) gene expression driven by the promoter of the vascular endothelial growth factor (VEGF) gene that is activated by hypoxia is effective in killing highly metastatic Lewis lung carcinoma A11 cells under hypoxic conditions. We isolated the promoter region encompassing the hypoxia response element (HRE) of the mouse VEGF gene. To assess the hypoxia responsiveness of the VEGF promoter, A11 cells were transiently transfected with luciferase reporter plasmids. Exposure of the transfectants to hypoxia resulted in a 2-3-fold induction of luciferase activity. Deletion of the HRE site abolished VEGF promoter activity under both normoxic and hypoxic conditions. We constructed a retroviral vector harboring the HSV-TK or green fluorescence protein (GFP) gene under the control of the VEGF promoter. A11 cells transfected with vector harboring the VEGF promoter fused to the HSV-TK gene [A11(HRE/TK) cells] were more sensitive to ganciclovir than cells transfected with the control vector harboring the VEGF promoter alone, and the sensitivity of the A11(HRE/TK) cells was increased by exposure to hypoxia followed by reoxygenation. Culturing A11 cells transfected with vector harboring the VEGF promoter fused to the GFP gene under hypoxic conditions resulted in an increase in the expression of GFP. Monitoring GFP expression and vascularity in the A11 transfectant tumors revealed up-regulation of GFP expression in poorly vascularized regions. Administration of ganciclovir to mice bearing s.c. tumors formed by A11(HRE/TK) cells resulted in regression of the tumors. These results suggest a possible application of the suicide gene driven by the VEGF promoter to cancer gene therapy that efficiently targets hypoxic tumor cells.

Overexpression of DAN gene product in normal rat fibroblasts causes a retardation of the entry into the S phase.

Differential screening-selected gene aberrative in neuroblastoma (DAN) gene (previously named N03 gene), whose expression is significantly reduced in transformed cells, has recently been demonstrated to have a tumor-suppressive activity in vitro. In order to investigate biological roles of DAN gene product in normal rat fibroblasts (3Y1), marker-selected transfectants that expressed the high amount of DAN gene product were generated from 3Y1 cell lines. These clones did not exhibit morphological changes compared with parental 3Y1 cells; however, they showed a decrease in growth rate and a remarkable reduction in saturation density. Cell cycle analysis revealed that the overexpression of DAN gene product causes the retardation of the entry into the S phase. These results suggest that DAN gene product may have an important role in regulation of the entry of cells into the S phase.

Deletion mapping on the short arm of chromosome 3 in squamous cell carcinoma and adenocarcinoma of the lung.

We examined loss of heterozygosity in 49 adenocarcinomas and 18 squamous cell carcinomas of the lung with 19 RFLP markers on the short arm of chromosome 3. Although no interstitial deletions were observed in any squamous cell carcinomas, interstitial or partial deletions were detected in 23 adenocarcinomas. Identification of two common regions of deletion in adenocarcinomas, at 3p21.3 and 3p14.1-21.1, suggested the presence of at least two tumor suppressor genes on 3p within the same regions commonly deleted in renal cell carcinomas. Correlation between the frequency of loss of heterozygosity on 3p and histopathological grade of adenocarcinoma also was observed. These results imply an etiological difference between two major types of non-small cell lung cancers, adenocarcinoma and squamous cell carcinoma.

Deletion of the COOH-terminal region of p73alpha enhances both its transactivation function and DNA-binding activity but inhibits induction of apoptosis in mammalian cells.

The candidate tumor suppressor p73 has a high sequence homology with p53 within the NH2-terminal transactivation domain, the sequence-specific DNA-binding region, and the oligomerization domain. However, p73alpha, which is most abundantly expressed in many tissues and cells among the alternatively spliced forms of p73, has an additional long COOH-terminal tail that might distinguish the function of p53 and p73alpha or other p73 splicing variants. To examine the functional role of the p73alpha COOH-terminal region, we generated a series of p73alpha truncation mutants including p73alpha(1-247) (retaining only a transactivation domain), p73alpha(1-427) (lacking the most COOH-terminal region including a SAM domain), and p73alpha(1-548) (deleting an extreme COOH-terminal region except a SAM domain). When transfected into COS cells, all of p73alpha, p73alpha(1-548), and p73alpha(1-427) localized in the cellular nucleus, whereas p73alpha(1-247) localized in both nucleus and cytoplasm. Intriguingly, when compared with p73alpha, both p73alpha(1-427) and p73alpha(1-548) showed a significant stimulation of the transcription of luciferase reporters harboring three p53-responsive promoters (p21(Waf1), Mdm2, and Bax) in p53-deficient SAOS-2 cells. Gel retardation assays showed that DNA-binding activity of p73alpha(1-427) and p73alpha(1-548) was increased as compared with that of the full-length p73alpha. However, the colony formation assays using SAOS-2 cells demonstrated that, contrary to p73alpha, transfection of p73alpha(1-427) or p73alpha(1-548) resulted in no significant reduction of the number of colonies. These suggest that the distal COOH-terminal region of p73alpha is a cis- or trans-acting regulatory domain and regulates its functions diversely.

Induction of immunity in peripheral tissues combined with intracerebral transplantation of interleukin-2-producing cells eliminates established brain tumors.

Cytokine gene therapy for the induction of potent immune responses against central nervous system tumors has proven to have significant potential. However, this strategy needs improvement in the process of antigen presentation and/or insufficient recruitment of immunocompetent cells to achieve successful eradication of established brain tumors. We investigated the therapeutic potential of induced systemic immunity in peripheral tissues combined with interleukin-2 (IL-2) production in the vicinity of brain tumors to treat established brain tumors. Sequential magnetic resonance image monitoring showed that the combinatory therapy consisting of intracerebral (i.c.) transplantation of IL-2-producing rat gliosarcoma 9L (9L/IL-2) cells and s.c. vaccination using irradiated 9L or 9L/IL-2 cells could cure 9L-bearing rats, whereas either the i.c. injection of 9L/IL-2 cells or the s.c. vaccination produced little or marginal antitumor effects, respectively. Xenogeneic murine neuroblastoma cells secreting IL-2 could substitute for 9L/IL-2 cells, producing significant antitumor effects in the vaccinated rats. Tumor-specific cytotoxic activity was induced in the vaccinated rats but not fully in the rats treated only with i.c. injection of 9L/IL-2 cells. Immunohistochemical analysis revealed that a number of CD4(+) and CD8(+) T cells infiltrated into the brain tumors which were treated with the combinatory therapy. The level of cell infiltration was similar to that found in s.c. 9L/IL-2 tumors which were subsequently rejected. In contrast, the brain tumors treated with either i.c. transplantation of 9L/IL-2 cells or the s.c. vaccination showed only moderate infiltration of T cells. The combinatory strategy, i.c. grafting of IL-2-producing cells, and s.c. immunization of irradiated whole tumor cell vaccine, is, thus, effective for recruiting activated T cells into the brain tumor site and could be a potential therapy for brain tumors.

Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers.

We have shown that a Mr 37,000 protein whose expression is strongly enhanced in human lung cancer tissues is the subunit of glyceraldehyde-3-phosphate (GAPDH, EC 1.2.1.12). We have isolated a GAPDH complementary DNA from human lung cancer cells and deduced the complete amino acid sequence of the encoded protein. The structure of the lung cancer GAPDH is identical to that of liver GAPDH. In addition, we have found that GAPDH mRNA expression is markedly increased in human lung cancer tissues. These results disclose a molecular basis of increased glycolysis in cancer cells and reveal an important role of energy creating reaction in cancer cell growth.