Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

A genome-specific repetitive DNA sequence from Oryza eichingeri: characterization,localization, and introgression to O. sativa

In the course of transferring the brown planthopper resistance from a diploid, CC-genome wild rice species, Oryza eichingeri (IRGC acc. 105159 and 105163), to the cultivated rice variety 02428, we have isolated many alien addition and introgression lines. The O. eichingeri chromatin in some of these lines has previously been identified using genomic in situ hybridization and molecular-marker analysis. Here we cloned a tandemly repetitive DNA sequence from O. eichingeri IRGC acc105163, and detected it in 25 introgression lines. This repetitive DNA sequence showed high specificity to the rice CC genome, but was absent from all the four tetraploid species with BBCC or CCDD genomes. The monomer in this repetitive DNA sequence is 325–366-bp long, with a copy number of about 5,000 per 1 C of the O. eichingeri genome, showing 88% homology to a repetitive DNA sequence isolated from Oryza officinalis (2n=2x=24, CC). Fluorescent in situ hybridization revealed 11 signals distributed over eight O. eichingeri chromosomes, mostly in terminal or subterminal regions. Received: 28 November 2000 / Accepted: 3 April 2001     

A repetitive and species-specific sequence as a tool for detecting the genome contribution in somatic hybrids of the genus Medicago

 A highly repeated sequence (C300) was cloned from Medicago coerulea and its organization in the M. sativa-coerulea-falcata complex, M. arborea, and three somatic hybrids involving M. sativa, was investigated. Southern-blot analysis revealed a tandemly repeated array and a species-specificity of the sequence to those species belonging to the complex. Various degrees of amplification of C300 were detected among the species of the complex and the outcome in the somatic hybrids was dependent on parental composition. Sequence analysis revealed strong homology (96%) of C300 with a clone (E180) previously isolated from M. sativa. As FISH analysis showed that C300 was dispersed along the chromosomes of Medicago spp., it should prove a valid tool for establishing the chromosome origin of somatic hybrids. Received: 14 April 1997 / Accepted: 18 April 1997     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum

A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.     

A novel repetitive sequence associated with the centromeric regions of Arabidopsis thaliana chromosomes

 The middle repetitive fraction of the Arabidopsis genome has been relatively poorly characterized. We describe here a novel repetitive sequence cloned in the plasmid mi167, and present in ∼90 copies in the genome of Arabidopsis thaliana ecotype Columbia. Hybridization analysis to physically mapped YAC clones representing Arabidopsis chromosome 4 revealed four mi167-hybridizing loci, all clustered near the centromere. No other loci were detected on YAC clones covering chromosome 4. In situ hybridisation experiments to Arabidopsis chromosome spreads showed that mi167-hybridizing sequences are clustered at the centromeric heterochromatin of all five chromosomes; on two chromosomes the hybridization appeared to be localised on one arm. Additional mi167-hybridizing loci were detected, one of which was adjacent to a non-centromeric, heterochromatic region. This work supports the idea that the majority of middle repetitive DNA in the Arabidopsis genome is clustered. It also adds to our understanding of the organization of the centromere of Arabidopsis chromosome 4. Received: 19 February 1996 / Accepted: 30 June 1996     

Role of macrophage-colony-stimulating factor in regulating the accumulation and phenotype of tumor-associated macrophages

 In order to better define the role played by tumor-cell-derived macrophage-colony-stimulating factor (M-CSF) in regulating the recruitment and phenotype of tumor-associated macrophages, Polyoma large T-transformed fibroblastoid cell lines, derived from M-CSF-deficient osteopetrotic op/op mice and their phenotypically normal op/+ littermate controls, were inoculated into SCID (severe combined immunodeficiency) recipients and both the proportion and phenotype of the macrophages present within the tumors generated were determined. The results obtained indicate that, although tumors derived from M-CSF-deficient and M-CSF-producing tumor cell inoculate contain a similar proportion of macrophages, the macrophages isolated from tumors lacking M-CSF appear morphologically less mature and express lower levels of interleukin 1β, tumor necrosis factor α and FcRγII mRNA. Taken together, these data suggest that, although M-CSF does not appear to play a critical role in determining the macrophage content of these tumors, it does play a role in modulating the phenotype, and potentially the functional activity of the macrophages present within the tumor microenvironment. Received: 30 August 1996 / Accepted: 7 February 1997     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes

 We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon γ (IFNγ) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFNγ and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFNγ secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to up-regulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor. Received: 30 January 1996 / Accepted: 22 March 1996     

A novel reverse-genetic approach (SIMF) identifies Mutator insertions in new Myb genes

We have developed a new strategy designated SIMF (Systematic Insertional Mutagenesis of Families), to identify DNA insertions in many members of a gene family simultaneously. This method requires only a short amino acid sequence conserved in all members of the family to make a degenerate oligonucleotide, and a sequence from the end of the DNA insertion. The SIMF strategy was successfully applied to the large maize R2R3 Myb family of regulatory genes, and Mutator insertions in several novel Myb genes were identified. Application of this technique to identify insertions in other large gene families could significantly decrease the effort involved in screening at the same time for insertions in all members of groups of genes that share a limited sequence identity. Received: 1 June 2000 / Accepted: 26 June 2000     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami : an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83 bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution. Received: 7 January 1997 / Accepted: 13 May 1997     

Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor &;b by treatment with bleomycin

 We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-β (TGFβ) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was signficiantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGFβ antibody. TGFβ activities were found in KDH-8-tumor-tissue-conditioned medium without acid treatment and were found in tumor-cell-conditioned medium after acid treatment; TGFβ mRNA and TGFβ protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor-derived TGFβ might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor-derived TGFβ and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significiant reduction of TGFβ activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGFβ activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGFβ. Received: 12 June 1995 / Accepted: 3 November 1995     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995