A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain.

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

Heterogeneous nuclear ribonucleoprotein B1 as a new marker of early detection for human lung cancers.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an RNA binding protein that is required for maturation of mRNA precursor. Tockman et al. previously reported that hnRNP A2/B1 with a M(r) of 31,000 is overexpressed from the early clinical stage of human lung cancer (M. S. Tockman et al., J. Clin. Oncol., 6: 1685-1693, 1988). However, when hnRNP A2/B1 mRNA and hnRNP B1 mRNA were separately studied, we found unique evidence that hnRNP B1 mRNA, which is a splicing variant of hnRNP A2 mRNA, was more significantly elevated in lung cancer tissues than hnRNP A2/B1 mRNA. Our hnRNP B1-specific polyclonal antibody specifically recognized hnRNP B1 protein as a M(r) 37,000 nuclear protein by Western blotting but did not recognize hnRNP A2 protein. Immunohistochemical staining with the hnRNP B1 antibody revealed that hnRNP B1 protein was specifically stained in the nuclei of human cancer cells, and in squamous cell carcinomas in particular, but not in those of normal adjacent lung epithelial cells. We think that hnRNP B1 protein of M(r) 37,000, not hnRNP A2, is well qualified as a biomarker for the detection of human lung cancer.

Consistent disruption of the AML1 gene occurs within a single intron in the t(8;21) chromosomal translocation.

The AML1 gene on chromosome 21 was rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia (AML). Southern blot analysis of 21 AML patients with t(8;21), including three with complex translocations, t(8;V;21), demonstrated that all the breakpoints occurred at random within a single intron between two coding exons of AML1. Clustering of the breakpoints in the restricted intron suggests the formation of a unique fusion gene between the AML1 gene and a presumable counterpart gene on chromosome 8. Nucleotide sequencing of the breakpoint region revealed that the translocation event was accompanied by deletion of a short stretch of nucleotides.

MTG8 proto-oncoprotein interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase in lymphocytes.

AML1-MTG8 chimeric oncogene is generated in acute myelogenous leukemia with t(8;21), and seems to be responsible for the pathogenesis of the disease. However, the role of MTG8 is ambiguous. Here we found that MTG8 interacted with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 was NHR3 domain, and that of RIIalpha was the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha were overlapped at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes.

Size difference in catalytic polypeptides of two active forms of mouse DNA polymerase alpha and separation of the primase subunit from one form, DNA replicase.

There are two active forms of DNA polymerase alpha in mouse cells. One form (DNA replicase) is a DNA polymerase associated with primase activity and the other form (7.3 S polymerase) has no primase activity (Yaugar, T., Kozu, T. and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127). The primase activity was dissociated from partially purified DNA replicase by hydroxyapatite column chromatography in buffer containing dimethyl sulfoxide and ethylene glycol. Nearly homogeneous primase, consisting of a 58 kDa polypeptide was obtained by glycerol gradient sedimentation and DEAE-cellulose column chromatography. Experiments on the effect of proteinase treatment and measurement of the molecular weight of the catalytic polypeptide of DNA replicase after its dissociation from the primase polypeptide indicated that the primase is not part of the DNA polymerase molecule, but an independent protein associated with DNA polymerase alpha, and that the latter is a 115 kDa catalytic polypeptide. The other form of DNA polymerase alpha, 7.3 S polymerase, consists of a 72 kDa catalytic polypeptide. Thus, the two forms of mouse DNA polymerase alpha have partially, if not completely, different catalytic polypeptide structures, suggesting that the 7.3 S polymerase is not simply formed from DNA replicase by dissociation of the primase subunit.

Significance of MTG8 in leukemogenesis.

MTG8 is a counterpart gene of AML1 in acute myeloid leukemia with t(8:21) translocation. Most of the coding region of the MTG8 is fused with AML1 runt domain. In normal tissues, the MTG8 is highly expressed in brain, but not in hematopoietic tissues. MTG8 may be important in leukemogenesis as well as in AML1 truncation. The function of MTG8 is assumed to be as a transcription factor, because it possesses several features common to transcription factors; putative zinc finger motifs, serine/threonine/proline-rich sequences and a region similar to TAF110. In this paper, we report on the protein properties of the MTG8.

In vitro catalytic activities of DNA/RNA chimeric hammerhead ribozymes against AML1-MTG8 mRNA, a fused gene transcript in acute myeloid leukemia with t(8;21).

In order to design the best construct for therapeutic hammerhead ribozymes against AML1-MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia, we synthesized DNA/RNA chimeric ribozymes directed to the area adjacent to the fusion point between AML1 and MTG8. Catalytic efficiency and fusion gene specificity of ribozymes were examined by kinetic studies of the cleavage reactions of AML1-MTG8, AML1, and MTG8 RNAs transcribed in vitro. Ribozyme 2 (Rz2) specifically cleaved AML1-MTG8 RNA at three nucleotides downstream of the fusion junction with high efficiency. The highest cleavage efficiency was achieved by Rz4.3, which targeted non-contiguous sequences and cleaved at 19 nucleotides downstream of the fusion junction. Rz4.3 also cleaved MTG8 RNA but the cleavage efficiency was three orders of magnitude lower than that for AML1-MTG8 RNA. Therefore, Rz4.3 and Rz2 are the proper ribozymes for in vivo application to modulate gene expression of the AML1-MTG8.

Designing of chimeric DNA/RNA hammerhead ribozymes to be targeted against AML1/MTG8 mRNA.

For therapeutic purposes, two chimeric DNA/RNA hammerhead ribozymes were synthesized to cleave AML1/MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia. One ribozyme, A/MRZ-1, recognizes the area adjacent to the fusion point between AML1 and MTG8, and cleaves six bases downstream from this point. The other, MRZ-1, recognizes the MTG8 sequence. Both ribozymes cleaved synthetic chimeric DNA/RNA substrates at theoretical sites. Neither cleaved AML1 RNA. A/MRZ-1 cleaved only AML1/MTG8 RNA, and MRZ-1 cleaved both AML1/MTG8 and MTG8 RNAs. The two ribozymes showed growth inhibition of an acute myeloid leukemia cell line carrying t(8;21), SKNO-1 cells. The same extent of growth inhibition was attained by antisense oligonucleotides against AML1/MTG8 RNA. The results suggest that the ribozyme has the potential to be developed as a useful agent for gene therapy, in particular for leukemia with t(8;21).

Expression of the tumor necrosis factor alpha gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes.

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor alpha gene (TNF alpha) and early-response genes in rat liver after its i.p. administration, and since TNF alpha had tumor-promoting activity in vitro, it is possible that TNF alpha itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of the TNF alpha gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly induced TNF alpha gene expression in rat hepatocytes, as well as TNF alpha release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of the TNF alpha gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 microM nodularin or microcystin-LR induced expression of the c-jun, jun B, jun D, c-fos, fos B and fra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that the TNF alpha gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.

Heterogeneous forms of poly(rA) . oligo(dT)-directed DNA polymerase activity from rat spleen.

Three forms of DNA polymerase, named enzymes A, B, and C, that preferred (rA)n x (dT)12-18 as a template-primer, were partially purified from an extract of rat spleen. Enzymes B and C, both sedimenting at 9S, appeared to correspond to DNA polymerase gamma. However, they differed in their behavior on phosphocellulose and DNA-cellulose column chromatographies, and in their optimum KCl and divalent cation requirements for activity. Enzyme A showed a unique property. Like DNA polymerase beta, it sedimented at 3.8S, was resistant to reagents blocking sulfhydryl groups, and was inhibited by phosphate, but it differed from DNA polymerase beta with respect to elution positions from DEAE-cellulose, phosphocellulose and DNA-cellulose columns, Km value (lower by one order of magnitude for dTTP), and template-primer preference. Enzyme A was found in the mitochondrial fraction, in which DNA polymerase beta was not detectable. Enzymes A and C were isolated from the nuclear fraction, but this fraction did not contain enzyme B. The cytosol contained only enzyme A. The mitochondrial fraction contained enzyme A and enzyme C-like polymerase. Enzyme B was obtained with enzymes A and C only by extraction of the whole cell homogenate. Enzyme B may be labile or may be an artificial form of DNA polymerase gamma formed during the purification procedures.

Inhibition of DNA polymerases alpha and gamma by rifamycin derivative AF/013.

The nature of the inhibitory effects of rifamycin derivative AF/013 (O-n-octyloxime of rifamycin SV) on DNA polymerases alpha and gamma were studied. Lineweaver-Burk analysis of the inhibition of DNA polymerases with respect to a substrate and template-primer showed a different mode of inhibition by AF/013 for each: the inhibition of DNA polymerase gamma was competitive with both dTTP and poly(rA)-oligo(dT), while that of DNA polymerase alpha was competitive with activated calf thymus DNA and non-competitive with dTTP. Further analysis of the competitive mode of the inhibition of DNA polymerase alpha, using poly(dT)-oligo(rA) as a template-primer, demonstrated that the primer molecule competed with AF/013. A change of effective divalent metal ion (Mn2+ in place of Mg2+) in the reaction mixture did not alter this competitive mode of inhibition with respect to the template-primer. The results of experiments to obtain further insight into the mechanism of drug-enzyme interaction suggest that AF/013 binds tightly to DNA polymerase alpha, and inhibits the process of chain elongation with DNA polymerases alpha and gamma.

Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization.

A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly(dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly(dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and alpha-amanitin (100 micrograms/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly(dT) and poly(dC) significantly more than RNA polymerases I and II or DNA polymerase alpha activity with poly(dT) . oligo(rA) and poly(dC) . oligo(dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies.

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.

Embryological fusion between the ducts of the ventral and dorsal primordia of the pancreas occurs in two manners.

The junction between the main pancreatic duct and the accessory duct has been thought to be the site of fusion between the ducts of the ventral and the dorsal primordia of the pancreas. The aim of this study was to investigate the fusion point between the ventral and the dorsal pancreatic ducts and to determine whether there is any relationship between the configuration of the pancreatic ducts and the manner of embryological fusion. Pancreatography was performed at 22 consecutive autopsies. Immunohistochemical staining of pancreatic polypeptide (PP) was performed because PP cells were rich in the ventral pancreas but poor in the dorsal pancreas. We identified two types of fusion. In one type, the ventral and the dorsal pancreatic ducts fuse at their junction (one-point fusion). In the other type, the two ducts fuse not only at the proximal site but at a second, more distal site (two-point fusion). Analysis of the pancreatograms showed that the distance between the junction and the major papilla in two-point fusion is significantly shorter than in one-point fusion (p < 0.01). These results indicate a close correlation between the pattern seen on pancreatograms and the manner of embryological fusion.

Pancreatic development and anatomical variation.

The pancreas is formed by the fusion of the ventral and dorsal anlage, and a wide spectrum of anomalies or anatomical variations may appear related to this complicated process of fusion: e.g., agenesis, aplasia of a pancreatic anlage, hypoplasia, annular pancreas, pancreas divisum or nonfusion of the ventral and dorsal duct system, pancreaticobiliary maljunction, etc. Every endoscopist who engages in pancreatography or related diagnostic and therapeutic procedures should always be aware of all sorts of anatomical variations he or she might encounter.

Cancer inhibition by green tea.

Green tea is now an acknowledged cancer preventive in Japan. This paper discusses several important features of (-)-epigallocatechin gallate (EGCG), the main constituent of green tea and tea polyphenols. EGCG and other tea polyphenols inhibited growth of human lung cancer cell line, PC-9 cells with G2/M arrest. 3H-EGCG administered by p.o. intubation into mouse stomach revealed that small amounts of 3H-activity were found in various organs where EGCG and green tea extract had previously demonstrated their anticarcinogenic effects, such as skin, stomach, duodenum, colon, liver, lung and pancreas. Cancer onset of patients who had consumed over 10 cups of green tea per day was 8.7 years later among females and 3.0 years later among males, compared with patients who had consumed under three cups per day. The mechanisms of action of EGCG were briefly discussed with regard to inhibition of tumor necrosis factor-alpha (TNF-alpha) release.

[Helicobacter pylori & gastric disease].

Since the discovery of Helicobacter pylori(H. pylori), causal linkage between H. pylori infection and some of gastric disease has been generally accepted from the results of many studies. Indeed the usefulness of H. pylori eradication therapy for acute gastritis, peptic ulcer, gastric polyp and MALT lymphoma etc. has been reported. In the low grade MALT lymphoma, the regression rate by this therapy is about 70%. On the other hand, we should pay the caution to several adverse effects, such as drug resistance and GERD, of H. pylori eradication therapy. However, based on the several results of comparative studies between antibiotic therapy and the other one, the antibiotic therapy for peptic ulcer is only covered by national health insurance at present. The reversibility of gastric precancerous conditions such as mucosal atrophy, intestinal metaplasia and dysplasia by antibiotic therapy has been studied, but its significance is not clear yet. In animal experiment, H. pylori infection induced gastric adenocarcinoma in Mongolian Gerbils. However, this phenomenon is limited to this kind of animal only. To proof the causal link between H. pylori infection and genesis of gastric cancer in human being, clinical intervention trials are ongoing in the world. If these trials can clarify it, the H. pylori eradication therapy will be established as preventive measure for gastric carcinogenesis.

[A cases of antithrombin III (ATIII) deficiency associated with extrahepatic portal occlusion undergoing operation for esophgogastric varices].

A 23-year-old woman developed thrombosis of the superior mesenteric vein and underwent an extensive enterectomy. She was diagnosed to have ATIII deficiency with extrahepatic portal vein thrombosis and esophagogastric varices. She was admitted to our department and underwent esophageal mucosal transection and splenectomy. Her activities of ATIII were 46%, but ATIII activities of her family were over 90%. ATIII activities during perioperative period were kept more than 70% following administration of ATIII drug. After splenectomy thrombocythemia which was over 300 x 10(4)/mm3 appeared with severe headache and slight pain of hands. She was discharged on 76th postoperative day with no complications and collapse of esophageal varices.

[Problems in therapy of thoracic esophageal cancer in view of recurrence in the lymph nodes].

The forms of recurrence from the first onset were confirmed in 171 out of 776 patients with thoracic esophageal cancer excised at our Department from 1959 to 1987; 87 patients (50.9%) had recurrence in the lymph nodes. Postoperative radiation in order to prevent recurrence in the lymph nodes was useful for the prevention of recurrence in the cervical lymph nodes, but radiation myelopathy/radiation pneumonitis might be of therapeutic difficulty in patients with recurrence in the areas of radiation. Moreover, patients treated by irradiation were apt to be involved in visceral recurrence. Incidence of recurrence in the lymph nodes was less in patients who had dissection in three areas than that in patients who received dissection in one or two. However, recurrence was observed in the border region between the cervix and the thorax, on the left side of the trachea, in the anterior portion and on the left side of the hilum in the areas of dissection. Useful postoperative chemotherapy is desirable in consideration of the fact that recurrence in the lymph nodes was observed at the posterior region of the pharynx, at the temporal region and in the pelvis and that dissemination and visceral recurrence were increased.

[A case of fatal graft-versus-host disease following blood transfusion in esophageal cancer documented by homozygous changes of HLA typing].

A 72 year-old Japanese male with esophageal cancer underwent esophagectomy. After seemingly uneventful recovery, he developed high fever on 11 post-operative day (POD), rashes over the whole body on 13 POD and leukopenia on 15 POD. On 22 POD, thrombopenia and parenchymal bleeding of lungs were noted. He died on 26 POD after progressive hypoxia and hypotension. HLA type of peripheral lymphocytes on him changed homozygously to that of the transfused fresh blood. Skin biopsy showed mild leukocyte infiltration in the epidermis and the dyskeratotic keratinocytes were associated with a contiguous lymphocyte, the so-called satellite cell necrosis. In the findings of autopsy, aplastic bone marrow and atrophied spleen, whose weight was 14g, were noted. Based on the clinical picture, skin biopsy and HLA study findings, we diagnosed this case as post-transfusion GVHD. We think that high age, operative injury and preoperative irradiation might be inducement to reveal post-transfusion GVHD in this case.