Identification of human autoantibodies to the transcriptional repressor ZF5.

ZF5 was originally cloned as a transcriptional repressor on the mouse c-myc promoter. It contains the Kruppel-type zinc fingers and a conserved POZ domain, which is found in a growing number of zinc finger proteins and mediate protein-protein interactions. Autoantibodies against transcription factors are sometimes found in sera from patients with high levels of anti-nuclear antibody (ANA). Using Western blotting with ZF5 and sera of autoimmune disease, we detected one serum, named M6 serum, which contains the antibody against a transcriptional repressor ZF5. The confirmed epitope was specific to ZF5 and was not reactive to the other analogous factors: BCL-6, ZID, and Sp1. This epitope also has a molecular mimicry of the viral proteins. From these results, we predict that viral protein which mimics host ZF5 antigen triggers self-reactive T cell clones and induces the autoantibody in M6 serum after the destruction of host tissues.

Analysis of the consensus binding sequence and the DNA-binding domain of ZF5.

Murine ZF5 is a transcription factor with five zinc finger motifs that represses the c-myc gene by binding to two GC-rich elements at the promoter region. Because of its ubiquitous expression in a variety of tissues, elucidation of biological functions and cellular target genes of ZF5 is of great interest. As the first step of identifying cellular target genes, we have attempted to determine the consensus binding motif for ZF5. We succeeded in isolating 19 oligonucleotide duplex DNAs to which ZF5 binds and determined the binding sequences with DNase I footprinting analysis. From these sequences, we deduced the consensus binding motif for ZF5 to be GSGCGCGR. In addition, we have analyzed the DNA-binding domain of ZF5 by testing a series of deletion mutants. It turned out that the zinc fingers 3 and 4 of the five finger motifs play a critical role in DNA binding.

ZF5, which is a Kruppel-type transcriptional repressor, requires the zinc finger domain for self-association.

ZF5, which we have cloned as a transcriptional repressor on the mouse c-myc promoter, has the POZ domain at the amino-terminus and the Kruppel-type zinc finger domain at the carboxy-terminus. In this report, we showed that ZF5 has two contradictory functions in transcription: activation of human immunodeficiency virus (HIV) promoter and repression of the HSV thymidine kinase (TK) promoter. The POZ domain contributed to the repressor activity, whereas the active function resulted from the DNA-binding ability of the zinc finger domain. We demonstrated that the POZ domain has a function mediating homomeric protein-protein interaction and this interaction requires the zinc finger domain. Furthermore, the POZ domain decreased the DNA-binding activity of the zinc finger domain. These results can provide evidence indicating the important interaction between the POZ and zinc finger domains.

Genomic cloning and characterization of the mouse POZ/zinc-finger protein ZF5.

We isolated genomic DNA containing the entire sequence of ZF5, which was originally identified by its ability to repress the mouse c-myc promoter and which was characterized as one of the POZ (Poxvirus and zinc finger) proteins. The POZ motif is a protein-protein interaction interface found at the N-terminal region of zinc finger proteins. Sequence analysis demonstrated that the ATG translation initiation codon was separately located from the remainder of the coding sequence. Using both RNase protection and primer extension assay, a single major transcription start site was determined. Promoter analysis by transient transfection assay suggested positive autoregulation by ZF5 itself. The ZF5 N-terminal region, including the POZ domain, was required for this regulation. Sp1 also activated the ZF5 promoter and this activity was repressed by addition of ZF5. ZF5 expression was stronger in mouse ovary, lung and brain than in other organs.

Detection of mouse skeletal muscle-specific product, which includes ZF5 zinc fingers and a VP16 acidic domain, by reverse transcriptase PCR.

ZF5, which we have cloned as a repressor on the mouse c-myc promoter, is a zinc finger protein containing Kruppel-type zinc finger and ZiN/POZ domains. In a reverse transcriptase PCR assay using mouse skeletal muscle RNA, we identified a 827 bp PCR product including the zinc finger domain of ZF5 and the acidic domain of VP16. The presence of the VP16 acidic domain induced the reduction of DNA-binding activity of the zinc finger domain. In addition, the inhibitory effect of the VP16 acidic domain was demonstrated on the human immunodeficiency virus (HIV) promoter, but there was no effect on the thymidine kinase (TK) promoter.

Establishment and characterization of a malignant epithelioid hemangioendothelioma from mouse thyroid tumor.

A mouse epithelioid hemangioendothelioma (mEHE) cell line, passagable in vivo without TSH stimulation, was established from a thyroid tumor. Before establishment of the cell line, the primary thyroid tumor which was co-transplanted with a TSH-producing pituitary adenoma showed signs of hyperplasia and then transformed during several in vivo transplantable passages. The cell line which was established from an in vitro culture, was deficient in thyroid-specific differentiation function and had characteristics of endothelial cell origin such as vascular cavity formation and factor VIII expression. The cell growth of mEHE/CH1-5 cell lines was independent of TSH but was inhibited by c-AMP and vitamin D3. c-Myc proto-oncogene expression was also suppressed by vitamin D3 treatment. Both serum depletion and heparin treatment induced morphological changes in mEHE/CH 5 from an epithelial cell-like to an endothelial cell-like shape. Immunohistochemical analysis showed that epithelial membrane antigen expression was decreased and factor VIII expression was increased in relation to the morphological changes. In a further in vivo transplantation experiment, the histology of the mEHE/CH5 cell tumor had an angiosarcoma-like structure. These results indicate that this cell line established from a thyroid tumor possesses both epithelial and endothelial characteristics. The mEHE/CH5 cells might provide a good model for analyzing thyroid tumorigenesis and allow functional characterization of endothelial cells.

Over-expressed ZF5 gene product, a c-myc-binding protein related to GL1-Kruppel protein, has a growth-suppressive activity in mouse cell lines.

ZF5 encodes a zinc finger protein, which contains five C2H2-type zinc fingers showing homology with the zinc finger of the Kruppel family, and binds to two sites in the mouse c-myc promoter. We report the effect of over-expression of ZF5 on cell growth. ZnCl2 treatment suppressed the growth of a mouse fibroblast cell line (L cells) transfected with the wild-type ZF5 gene driven by the metallothionein promoter. Cells transfected with the wild-type ZF5 gene formed colonies two- to fivefold less efficiently than those transfected with the mutant ZF5 gene in P19, NIH3T3, 3T3-L1 and L cells. Over-expression of ZF5 did not cause c-myc down-regulation or arrest of the cell cycle, but increased the DNA content.

Blocking of DNA synthesis in vitro by a guanosine 2',3'-cyclic phosphate: a possible mechanism of chromosome aberrations induced by U5 snRNA.

U5 snRNA can induce both transformation and chromosome aberrations of cells. The polypurine tract, GGAGAGGAA, of the RNA has been suggested to participate in both phenomena. In vitro transcription expected to give this polypurine oligoribonucleotide was associated with cleavage of transcripts, generating 5'-terminal hydroxyl and 3'-terminal 2',3'-cyclic phosphate groups. The cleavage was further studied by making use of a Mg(2+)-catalyzed reaction and RNase T1 and RNase U2 digestion. The cleavage was found to generate highly reactive RNA molecules, participating in subsequent ligation of RNAs. Such a reactive molecule, guanosine-2',3'-cyclic phosphate, was capable of blocking DNA synthesis in vitro. The results may provide a possible mechanism of the chromosome aberrations induced by U5.

Random process of metastasis and generation of heterogeneity in a mouse sarcoma line.

The process of metastasis was analyzed in 505-05-01 cells, an established line of a methyl-cholanthrene-induced mouse sarcoma, by tagging the cells with the pSV2neo plasmid. A dominant clone was identified which, upon transplantation together with other clones, overgrew tumors in the kidney capsule. However, when this clone was transplanted in a mouse collaterally with recessive clones, both clones grew at the same rate and metastasized to the lung at an equal frequency. This suggests that the process of metastasis in this particular sarcoma line is stochastic, the dominant population having a better chance to colonize to the lung. The dominant neomycin-resistant clone was transfected with another marker plasmid, pY3, which confers resistance to hygromycin. Results of mixed inoculation of 9 independently isolated clones revealed the hierarchy of dominance among clones. This indicated the existence of heterogeneity within the parental clone. Upon mixed inoculation with hygromycin-resistant clones, the parental clone overgrew in the tumors. This indicated that some clone had changed its phenotype to become less aggressive. Thus, the direction of phenotypic drift in vitro seems to be random in terms of behavior in vivo.

Carcinogenic effect of in utero 252Cf and 60Co irradiation in C57BL/6N x C3H/He F1 (B6C3F1) mice.

C57BL/6N x C3H/He F1 mice were exposed in utero to 0, 1.0 and 2.7 Gy of 252Cf or 60Co at day 16.5th of gestation. Mice of both sexes were observed for 2 years. The females in the irradiated groups showed increases in the incidences of pituitary, mammary gland, liver and lung tumors. 252Cf was more effective in inducing tumors than was 60Co. Interestingly, the incidence of hematopoietic tumors decreased by irradiations with 252Cf but not with 60Co. The incidences of liver tumors in males increased by 252Cf-irradiation, whereas, the incidences of skin and soft tissue tumors increased by 60Co-irradiation. These results indicate that irradiation in utero during the late embryonic stage can induce tumors postnatally after a long latency. Moreover, females irradiated in utero had disfunction of the ovaries, evidence of impairment of the female's specific hormonal environment. This may be the cause of the low incidence of ovarian tumors and the high incidences of liver, lung and pituitary tumors in these female mice. Females with pituitary tumors had a high serum prolactin, which might be responsible for the concurrence of mammary gland tumors. These results indicate the importance of host factors in the development of radiation-induced tumors.

Somatic mutation during metastasis of a mouse fibrosarcoma line detected by DNA fingerprint analysis.

Metastatic nodules were examined by DNA fingerprint analysis. The probes used, Pc-1 and Pc-2, detect mutations as shifts in bands of the minisatellite loci which are dispersed among chromosomes. Four clonal lines of a fibrosarcoma from an F1 mouse (C57BL/Ka x C3H/He) were selected for various metastatic potentials upon inoculation into syngeneic mice. These four lines exhibited many extra bands resulting from recombination and/or DNA slippage, indicating accumulation of mutations during the successive passages in mice. One of the four, a 505 cell line which had been passaged extensively in vitro and consisted of a heterogenous population, was inoculated into thirteen syngeneic mice, and gave rise to six lung metastatic nodules in two mice. All the nodules showed band-patterns distinct from one another, although nodules within a given mouse tended to show similar patterns. When a genetically tagged 505-05-01 clone was analyzed, three of nine metastatic nodules obtained also revealed new bands. These results strongly suggest that somatic mutations occur at a high frequency during metastasis, providing direct evidence of genetic instability of the tumor cells.

Effect of the administration of bismuth nitrate on radiogenic thymoma induction in mice.

Metallothionein functions as a radical scavenger protecting cells from the indirect effect of radiations. We investigated the effect of bismuth nitrate, an efficient inducer of metallothionein, on acute and late effects of radiation in mice. Metallothionein contents were examined in several organs after the administration of bismuth nitrate. The content in bone marrow increased 2-fold in the treated as compared to the control mice. This treatment protected irradiated mice from bone marrow death and increased the number of endogenous spleen colonies. The metallothionein content in the ileum did not change after treatment with bismuth nitrate. Mice were not protected by bismuth nitrate when exposed to 9 Gy of X-rays. This suggests that this agent does not protect from gastrointestinal death. The incidence of X-ray-induced thymic lymphomas was lowered by the administration of bismuth nitrate in mice exposed to four fractionated doses of 1.3 Gy of X-rays. These results indicate that bismuth nitrate effectively modified both acute and late effects of X-rays by inducing metallothionein in the target tissues.

Carcinogenic effects of tritiated water (HTO) in mice: in comparison to those of neutrons and gamma-rays.

A series of experiments was carried out using female (C57BL/6N x C3H/He)F1 mice (BCF1) to assess the carcinogenic effect of tritiated water (HTO) following a preliminary experiment in which the acute effect of HTO was studied in young female mice of C57BL/6L strain in an attempt to gain basic information on the long-term experiment, and the results were compared with those of fission spectrum neutrons and gamma-rays. The obtained findings are summarized as follows. 1) C57BL/6N mice receiving a single intraperitoneal (i.p.) injection of HTO, 7.4 x 10(8) Bq (20 mCi) or more, died of bone marrow failure within 20 days. 2) In long-term experiment, BCF1 mice given 1.4 x 10(8) Bq to 5.6 x 10(8) Bq developed solid tumors in a variety of tissues but with no obvious dose dependency. 3) The fractionated exposure of 7.4 x 10(8) Bq (1.9 x 10(8) each, at 7-day intervals) was highly effective in induction of malignant T-cell lymphomas (85%) with a shorter latency than that of a single exposure (15%). 4) A protracted irradiation of 0.27 Gy of 137Cs, designed to simulate the decreasing absorption rate with time after a single i.p. injection of an equivalent dose of HTO resulted in a drastic reduction in induction rate of ovarian tumors as compared with that of an acute 60Co gamma irradiation. 5) The carcinogenic potential of HTO, given as a single i.p. injection, was quite similar to that of acute 60Co gamma ray irradiation at both 2.7 Gy and 0.27 Gy levels. On the other hand, the effect of HTO was slightly higher than that of protracted gamma-ray irradiation. 6) 252Cf fission neutrons was found to be more potent in tumor induction than gamma-rays or HTO beta-rays under the present experimental conditions.

Control of ductal vs. alveolar differentiation of mammary clonogens and susceptibility to radiation-induced mammary cancer.

We have developed an in vitro-in vivo transplantation assay for measuring the concentration of clonogenic epithelial cells in cell suspensions of rat mammary tissue. Rat mammary clonogens from organoid cultures are capable of the same degree of PLDR as clonogens in vivo. The growth and differentiation of mammary clonogens to alveolar colonies or ductal colonies is regulated as follows: a) in the presence of E2 and high prolactin (Prl), cortisol induces mammary clonogens to proliferate and differentiate to form alveolar colonies which secrete milk and begin losing clonogenic potential, b) in cortisol deficient rats, Prl and E2 synergistically stimulate non-secretory ductal colonies, formation of which retain clonogenic potential, c) E2 without progesterone stimulates alveolar colony formation in the presence of cortisol and high Prl, d) progesterone inhibits mammary clonogen differentiation to milk-producing cells and induces ductogenesis in a dose responsive fashion in the presence of E2, cortisol and high Prl. High prolactin levels coupled with glucocorticoid deficiency increases the susceptibility to mammary carcinogenesis following low dose radiation exposure by increasing the number of total mammary clonogens which are the presumptive target cells and by stimulating their proliferation after exposure.

Analysis of transforming genes in indirectly induced radiogenic thymomas in mice.

The expression of oncogenes was studied in 12 types of 178 mouse tumors induced by radiations and chemicals. DNA was analyzed in tumors in which the overexpression of oncogenes was noted. Amplification of the myc oncogene was found in chemically induced sarcomas, but not in sarcomas induced by radiation. Activation of oncogenes by small mutations and the inactivation of tumor suppressor genes has to be taken in account in the radiation induction of mouse tumors. We therefore made further analyses of radiogenic thymomas. Loss of heterozygocity was revealed in directly induced thymomas by the deletions of allele specific minisatellite bands. Analysis of a hypervariable minisatellite locus also revealed that these thymoma cells suffered high recombinogenic activity during tumorigenesis. In addition, transfection of cellular DNA to normal Golden hamster cells identified the activated K-ras oncogene in the directly induced radiogenic thymomas. Indirectly induced radiogenic thymomas were tested similarly. Transformed cells from secondary transfection experiment were positive for the mouse-specific repetitious sequences, but devoid of mouse ras oncogenes. Indirectly induced radiogenic thymomas originate from unirradiated normal thymus cells transplanted in irradiated hosts. The spontaneous activation of oncogenes yet to be identified may therefore be involved in the development of this tumor.

Molecular species of phospholipid in rats in primary and transplanted fibrosarcomas induced by soybean oil containing tocopherol acetate.

When soybean oil containing tocopherol acetate was given to rats once a week subcutaneously for 10-12 months, it caused the development of fibrosarcomas at the injection site in 11 of 15 rats. A tumor produced in this manner proved eminently transplantable into other rats. The molecular species of phospholipid subclasses were determined in primary and transplanted tumors. The molecular species composition of the phospholipid subclasses in both types of tumors were similar. The percentages of diacyl and alkylacyl glycerophosphocholine (GPC) were 90-93 and 6-8% of total phosphatidylcholine, respectively. The percentages of diacyl and alkenylacyl glycerophosphoethanolamine (GPE) were 51 and 45%, respectively, of total phosphatidylethanolamine (PE). Diacyl and alkylacyl GPC species containing arachidonic acid (20:4) composed about 15-16 and 37-40% of each subclass, respectively. Diacyl and alkenylacyl GPE species containing 20:4 composed about 38-40 and 56-60% of each subclass, respectively. Disaturated species of diacyl and alkylacyl GPC composed about 22-24 and 13% of each subclass, respectively, whereas these species of PE composed less than 2%. The fatty acid composition of the other tumor phospholipids was analyzed.

Parathyroid tumors in atomic bomb survivors in Hiroshima: epidemiological study from registered cases at Hiroshima Prefecture Tumor Tissue Registry, 1974-1987.

An examination of registered cases of parathyroid tumor in Hiroshima Prefecture between 1974 and 1987 revealed 23 cases. An epidemiological study showed that the incidence of parathyroid tumors in Hiroshima Prefecture was significantly higher in the total exposed, especially among the proximally exposed (within 2,000 m from the hypocenter), than in the control nonexposed group (P less than 0.001). A similar trend was seen for parathyroid tumor associated with primary hyperparathyroidism.

Analysis of the binding proteins and activity of the long terminal repeat of Moloney murine leukemia virus during differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cell lines were established which carry the stably integrated chloramphenicol acetyltransferase (CAT) gene under the control of the transcriptional elements of the long terminal repeat (LTR) of Moloney murine leukemia virus. The activity of three elements of the stably integrated LTR was analyzed in undifferentiated EC cells (stable CAT assay). Results of the study are summarized as follows. (i) In the stable assay, the promoter region of the LTR was inactive in undifferentiated ECA2 and F9 cells, and the level of the activity was 10(-4) of that in NIH 3T3 cells. (ii) In contrast to the results of the transient assay, the enhancer was active in undifferentiated ECA2 cells and in F9 cells. It activated CAT activity more than 60-fold and about 8-fold in ECA2 cells and F9 cells, respectively. (iii) Suppression by ELP, the embryonal LTR-binding protein, was more pronounced in the stable assay than in the transient assay. These data suggest that, when compared with NIH 3T3 cells, a major factor for the inactivity of the LTR in EC cells is the inefficiency of the promoter in this assay. Transcriptional activity of the LTR was analyzed during the differentiation of EC cells. In the case of ECA2 cells, the magnitude of activation by the enhancer did not change during differentiation. The activity of the promoter increased about 10-fold, and the suppression by ELP became negligible 4 days after the induction of differentiation. Upon differentiation of F9 cells, the activity of the enhancer increased more than 300-fold, but the promoter remained inactive. The pattern of LTR-binding proteins also varied during the differentiation of EC cells. Our present data suggest that the activity of LTR elements as assayed by the stable assay differs from the activity as assayed by the transient assay. It also indicates that the activity of these elements exhibits cell-type-specific changes during the differentiation of EC cells.

Parathyroid tumors in atomic bomb survivors in Hiroshima: first report of surgical cases, 1956-1988.

Seventeen patients with parathyroid gland tumors underwent surgical resection at the Hiroshima University Hospital between 1956 and 1988. Three of them where born after the atomic bomb explosion, and 6 of the remaining 14 patients (42.9% of the total- a high proportion) were atomic bomb survivors. Because parathyroid gland tumors and hyperparathyroidism are assumed to develop in patients who have been exposed to radiation after a long latent period, it is necessary to anticipate the possible occurrence of these diseases in atomic bomb survivors. The present paper is the first to report surgically extirpated parathyroid gland tumors in atomic bomb survivors.

Induction of transplantable tumors by repeated subcutaneous injections of natural and synthetic vitamin E in mice and rats.

Natural vitamin E and synthetic vitamin E (dl-alpha-tocopheryl acetate) were tested for their tumorigenicity in rodents. Transplantable tumors, at the site of injection, were induced by repeated injections of these compounds in two strains of mice, NFS/N and C57BL/6N x C3H/He F1, and in a strain of rats, Fischer 344. Natural vitamin E was tumorigenic in both strains of female mice only when injected with soya oil. In contrast, dl-alpha-tocopheryl acetate alone was capable of inducing tumors in Fischer 344 rats. Only one out of 5 male NFS/N mice given dl-alpha-tocopheryl acetate developed a tumor. Therefore, Fischer 344 rats were more susceptible to tumor formation by dl-alpha-tocopheryl acetate than NFS/N mice. dl-alpha-Tocopheryl acetate with soya oil or with palm oil also resulted in the formation of transplantable tumors in NFS/N mice and Fischer 344 rats. There was no difference in the tumor incidence between mice treated with dl-alpha-tocopheryl acetate alone and dl-alpha-tocopheryl acetate plus soya oil or palm oil. However, in rats, the incidence was lower for a group treated with dl-alpha-tocopheryl acetate plus palm oil than for those with dl-alpha-tocopheryl acetate alone and with dl-alpha-tocopheryl acetate plus soya oil.