Xeroderma pigmentosum patients from Germany: repair capacity of 45 XP fibroblast strains of the Mannheim XP Collection as measured by colony-forming ability and unscheduled DNA synthesis following treatment with methyl methanesulfonate and N-methyl-N-nitrosourea

Summary A total of 45 XP fibroblast strains from the Mannheim XP Collection (representatives of XP complementation groups A, C, D, E, F or G, I, and XP variants) were investigated for colony-forming ability (term: D₀ after treatment with up to ten doses of the methylating carcinogen MeSO₂OMe. As controls 16 fibroblast strains from normal donors were used. Except for 4 XP strains (1 from group C and 3 from group D) which, however, were borderline cases, none of the remaining 41 XP strains was found to be more sensitive than normal controls. This held true within the limits of an experimental accuracy (experimental variability of D₀ values) of ±7%. When weighted means were calculated for XP complementation groups and compared with that of normal donors at a significance level of 5%, no significant difference was detected. In contrast, after exposure of 6 XP group D strains to MeNOUr, a weighted mean D₀ value was obtained which was significantly decreasd by 27%. Unscheduled DNA synthesis (term: G₀ which serves as a measure of excision repair) after exposure to MeNOUr was quantitatively the same (exposure to MeNOUr was quantitatively the same (experimental varability: ±8%) both in the group of normal strains and in most of the XP complementation groups. Exceptions were group E and group F (or G) which had higher, and group I which had lower repair. Analogous G₀ values measured after exposure to MeSO₂OMe (experimental variability: ±13%), however, differed from that of the control strains: they were lower in XP complementation groups A, D, E, F (or G), and I. However, groups A, E, F (or G), and I including only 3 individual strains or less may be considered to be possibly ill-represented. Yet, group D including 11 XP strains did show reduction of the mean G₀ value by 35%. From this it is concluded that there are repair defects in XP group D strains with regrad to MeSO₂OMe-induced adducts. These defects seem to be small.Abbreviations XP xeroderma pigmentosum - MeSO₂OMe methyl methanesulfonate - MeNOUr N-methyl-N-nitrosourea - Me(NO)(NO₂)Gdn N-methyl-N-nitro-N-nitrosoguanidine - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Xeroderma pigmentosum patients from the federal Republic of Germany: Decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity

Summary A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the UV-like carcinogen (Ac)₂ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D₀) was calculated for each cell strain.For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E₀.Comparing D₀ with E₀ values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previouly shown (Fischer et al. 1982) that D₀ values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA.A considerable similarity in sensitivity towards both UV light and (Ac)₂ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV-and (Ac)₂ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - UV light ultraviolet light - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Suppressor of yeast mitochondrial ochre mutations that maps in or near the 15S ribosomal RNA gene of mtDNA.

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho- strains of yeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSU1-carrying rho- clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho- does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.     

Diethylnitrosamine- and partial hepatectomy-induced decrease in α2u-globulin mRNA level in the rat liver

Changes in gene expression in the rat liver were investigated by analyzing cDNA libraries for liver mRNAs from adult male rats injected with a chemical carcinogen diethylnitrosamine (DEN). Differential screening using normal and DEN-treated liver cDNAs as probes demonstrated that some of the mRNA species had noticeably lower abundance in the DEN-treated liver than in the untreated liver. Surprisingly, most of those clones were found to code for _2u-globulin (A2uG), an abundant protein in the male rat liver. Further analysis by in situ hybridization revealed that the decrease in the A2uG mRNA level occurred in the area where liver cells were proliferating due to DEN treatment and/or partial hepatectomy (PH). The findings indicate coincidence of cell proliferation with a decrease in the A2uG gene expression in the adult male rat liver, implying that the A2uG-related change favors chemical carcinogen-induced cell growth.Abbreviations A2uG _2u-globulin - DEN diethylnitrosamine - PH partial hepatectomy     

Biochemical mechanisms on species differences in gastric carcinogenesis

Summary The biochemical denitrosation of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in tissues from four strains of rat, inbred Buffalo, Lewis, B-N, and the random-bred Sprague-Dawley, with different sensitivities to MNNG-induced gastric carcinomas was investigated as a possible explanation for the species/strain differences in MNNG-induced carcinogenesis. An analytical HPLC method was developed to assay denitrosation of MNNG to N-methyl-N-nitroguanidine (MNG) by cytosolic, microsomal, mitochondrial, and nuclear cell fractions. All the activity was contained in the microsomal and cytosolic fractions, with the major portion occurring in the cytosol. The activity in both fractions was NADPH-dependent, but denitrosation was not reduced by inhibitors of the cytochrome P-450 system. Denitrosation of MNNG post-mitochondrial supernatant (S9) fractions from liver, glandular stomach mucosa, and duodenal mucosa of the four rat strains was determined. In all strains, denitrosation activities were highest in liver. Comparisons between the three strains most sensitive to MNNG-induced gastric carcinogenesis indicated no large differences for any tissue. However, Buffalo, the most resistant strain, did have a higher level of denitrosating activity in all three tissues, which is consistent with the hypothesis that higher levels of detoxifying enzymes may lead to a decreased incidence of tumors. On the other hand, denitrosation accounts for less than 3% of the MNNG that disappears during the incubation perod so that the relevance of denitrosation as a mechanism in strain-specific sensitivity to MNNG-induced gastric carcinoma requires additional studies.Postdoctorate Fellow, American Health FoundationDedicated to Professor Hermann Druckrey, on the occasion of his 80th birthdaySupported by Public Health Service grant CA-30658 from the National Cancer InstitutePostdoctorate Fellow, American Health Foundation     

New tumor necrosis factor-α-inducing protein released from<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> for gastric cancer progression

Purpose To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor- (TNF-)-inducing protein of H. pylori was studied.Methods The HP0596 gene of H. pylori was identified as the TNF--inducing protein (Tip) gene from genome sequence of H. pylori strain 26695. Using recombinant Tip (rTip) and deleted Tip (rdel-Tip) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF- gene expression and NF-B activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells.Results Tip protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTip significantly induced TNF- gene expression and NF-B activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tip did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-B p65, and abrogated TNF- induction induced by Tip protein.Conclusion Tip is a new carcinogenic factor released from H. pylori mediated through NF-B activation.     

6-Methylguanine and 6-methylguanosine inhibit colony-forming ability in a malignant xeroderma pigmentosum cell line but not in other xeroderma pigmentosum and normal human fibroblast strains after treatment with 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea

Summary The XP cell strain XP29MA, its malignant counterpart XP29MAmal and a normal human fibroblast strain were tested for colony-forming ability after treatment with HECNU in the presence of m⁶G, m⁶Gua, and he⁷G.In XP29MAmal, inhibition of post-HECNU colony-forming ability was 35% when 0.25 mM of either m⁶G or m⁶Gua were present, whereas in XP29MA and the normal fibroblast strain no inhibition was detected. The he⁷G caused a similar but smaller inhibitory effect in XP29MAmal, but failed to do so in XP29MA.HECNU predominantly exerts its killing effect by alkylating O-6 of DNA-bound guanine and causing DNA interstrand crosslinks. Alkylation of O-6 of guanine can be repaired by 6-methylguanine-DNA methyltransferase. From our experiments we conclude that in XP29MAmal this methyltransferase was inhibited in the presence of the 6-alkylguanines, thus leaving more 2-chloroethylated sites in DNA unrepaired. This results in sensitization in terms of decreased colony-forming ability observed only in the malignant cell line.Abbreviations XP xeroderma pigmentosum - HECNU 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)-urea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - m⁶G 6-methylguanosine - m⁶Gua 6-methylguanine - he⁷G 7-(2-hydroxyethyl)-guanosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136The publication is dedicated to Professor E. Hecker on the occasion of his 60th birthday     

Comparison of DNA-incising capacities in fibroblast strains from the Mannheim XP collection after treatment with N-acetoxy-2-acetylaminofluorene and UV light

Summary The DNA-incising capacity was determined in 8 normal and 23 XP fibroblast strains of the Mannheim XP collection using the alkaline elution technique after treatment with both UV light and the UV-like carcinogen (Ac)₂ONFln. Experimental conditions were chosen to allow for selective monitoring of repair-specific enzyme-catalyzed breaks. In order to compare DNA-incising capacities of the various cell strains after UV irradiation with those after treatment with (Ac)₂ONFln, dose-response experiments including up to 8 dose levels were performed. The elution curves were analyzed by linear regression analysis. Elution velocities (in terms of DNA singlestrand breaks per 10⁶ nucleotides) were plotted against the square root of the doses. The slope of the resulting regression line yielded a characteristic term, designated E ₀, for the DNA-incising capacity of each cell strain. In contrast to normal fibroblasts, E ₀ was found to be reduced in all XP cell strains belonging to the complementation groups A, C, D, E, F (or G) and I investigated, after treatment with both UV light or (Ac)₂ONFln. Surprisingly, XP variant strains also exhibited lower E ₀ values. A comparison of post-UV with post-(Ac)₂ONFln DNA-incising capacities revealed that reduction in the E ₀ values was very similar in all XP cell strains tested. These data suggest that the sensitivity of XP cells towards UV light or (Ac)₂ONFln is due to the same enzymatic defect, namely impaired incision of DNA containing pyrimidine dimers or (Ac)₂ONFln-DNA adducts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine Dedicated to Prof. W. Kunz on the occasion of his 65th birthdayThis work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

The effects of inhibitors of topoisomerase II and quinacrine on ultraviolet-light-induced DNA incision in normal and xeroderma pigmentosum fibroblasts

Summary The aim of our work was to investigate whether DNA topoisomerase II participates in the repair-specific incision of UV-irradiated genomic DNA. Therefore, the influence upon DNA incision of the topoisomerase II inhibitors (nalidixic and oxolinic acid, novobiocin and coumermycin A₁) as well as the intercalating agent quinacrine has been measured in normal human fibroblasts using the alkaline elution technique. In addition, inhibition by novobiocin has been determined in fibroblast strains from 11 normal donors and from 16 xeroderma pigmentosum (XP) patients belonging to the complementation groups A, C, D, E, and XP variant. Nalidixic and oxolonic acid did not inhibit endonucleolytic cleavage, whereas novobiocin was a potent inhibitor of DNA incision. It was observed that in normal and in all XP strains 50% inhibition by novobiocin occurred on average in the dose range 315–590 M. Since inhibition by novobiocin was not paralleled by that with the other topoisomerase II inhibitors nalidixic and oxolinic acid, it must be concluded that reduction of enzyme-catalysed breaks was not due to the participation of topoisomerase II in the incision step, but to the displacement of ATP at the binding site of the DNA-incising enzyme. This enzyme absolutely requires ATP as a cofactor for endonucleolytic cleavage. Quinacrine, however, inhibited DNA incision in normal fibroblasts at a meanK _i of 318 M. Inhibition by this intercalating agent seems to be caused by structural perturbations in DNA, which render it a poor substrate for endonucleolytic cleavage.Abbreviations XP xeroderma pigmentosum - UV ultraviolet light This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the subtractive probes generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, aBLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na⁺, K⁺-ATPase -subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a -glutathioneS-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.This work was supported by research grants from the chinese National 8th 5-Year medical Strategic Science and technology Plan (85-914-01-09), the National Science foundation of China (39170800 and 39340007), and the Zhejiang Provincial Science Foundation (393041)     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)     

Comparison of sea urchin and human mtDNA: evolutionary rearrangement.

Clones of full-length mtDNA have been isolated from a Strongylocentrotus franciscanus recombinant DNA library by screening a cDNA clone of cytochrome oxidase subunit 1 mRNA. Restriction fragment cross-hybridization analysis shows the following difference in gene arrangement between sea urchin and human mtDNA. The 16S rRNA and cytochrome oxidase subunit 1 genes are directly adjacent in sea urchin mtDNA. These two genes are separated in human and other mammalian mtDNAs by the region containing unidentified reading frames 1 and 2. In spite of the difference in gene order, gene polarity appears to have been conserved. We conclude that the difference in gene order reflects a rearrangement that took place in the sea urchin lineage since sea urchins and mammals last shared a common ancestor.     

Overexpression of Gsα subunit in thyroid tumors bearing a mutated Gsα gene

Point mutations occurring at codon 201 of the gene coding for the subunit of the stimulatory G protein impair intrinsic GTPase activity and lead to a constitutive activation of adenylate cyclase. We have examined thyroid follicular and papillary carcinomas and follicular adenomas and found samples that bear this mutation at codon 201 of the Gs gene. Both mutation-positive and mutation-negative tissue samples were investigated for the level of Gs expression relative to a pool of normal thyroid tissue, using immunoblotting against two (mid-region-specific and C-end-specific) antipeptide antibodies. Using 8000g and 100 000g membrane fractions of homogenized tissues we have demonstrated that the Gs proteins in normal ad neoplastic thyroid tissues are represented by three isoforms: 43 kDa, 45 kDa and 52 kDa. We have quantified and compared the amount of Gs protein and find it is overexpressed in mutation-bearing tissue samples.Abbreviations Gs protein Stimulatory heterotrimeric guanine-nucleotide-binding protein - BCIP 5-bromo-4-chloro-3-indoxyl phosphate     

Mesoderm induction and blood island formation by angiogenic growth factors and embryonic inducing factors

Summary Factors which induce mesoderm, including endothelium lined cavities and primitive blood cells in omnipotent amphibian ectoderm, have been isolated from different sources. Recently it was shown that angiogenic factors, which belong to the protein families of the heparin binding growth factors (acidic and basic fibroblast growth factor) and the transforming growth factors (TGF- 1 and - 2), also induce mesodermal tissues in amphibian ectoderm. In Triturus ectoderm, capillary like endothelial networks are induced preferentially by the transforming growth factors. The relationship between growth factors and inducing fators is discussed.Supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie     

DNA variation and symbiotic associations in phenotypically diverse sea urchin Strongylocentrotus intermedius

Strongylocentrotus intermedius (A. Agassiz, 1863) is an economically important sea urchin inhabiting the northwest Pacific region of Asia. The northern Primorye (Sea of Japan) populations of S. intermedius consist of two sympatric morphological forms, "usual" (U) and "gray" (G). The two forms are significantly different in morphology and preferred bathymetric distribution, the G form prevailing in deeper-water settlements. We have analyzed the genetic composition of the S. intermedius forms using the nucleotide sequences of the mitochondrial gene encoding the cytochrome c oxidase subunit I and the nuclear gene encoding bindin to evaluate the possibility of cryptic species within S. intermedius. We have examined the presence of symbiont microorganisms by means of 16S rRNA sequences. The nucleotide sequence divergence between the morphological forms is low: 0.74% and 0.70% for cytochrome c oxidase subunit I and nuclear gene encoding bindin, respectively, which is significantly below average intrageneric sequence divergence among Strongylocentrotus species. We thus have found no genetic evidence of cryptic species within S. intermedius. Phylogenetic analysis shows that the bacteria symbionts of S. intermedius belong to the phylum Bacteroidetes, but the U and G forms predominantly harbor highly divergent bacterial lineages belonging to two different taxonomic classes, Flavobacteria and Sphingobacteria. We propose that the U and G forms of S. intermedius represent distinct ecomorphological adaptations to contrasting shallow- and deep-water marine environments and might be considered incipient species. We also propose that the symbiotic bacteria likely play an important role in the evolution of morphological divergence of S. intermedius.     

Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b.

Cytochrome b comprising 91-kDa and 22-kDa subunits is a critical component of the membrane-bound oxidase of phagocytes that generates superoxide. This important microbicidal system is impaired in inherited disorders known as chronic granulomatous disease (CGD). Previously we determined the sequence of the larger subunit from the cDNA of the CGD gene, the X chromosome locus affected in "X-linked" CGD. To complete the primary structure of the cytochrome b and to assess expression of the smaller subunit, we isolated cDNA clones for the 22-kDa polypeptide by immunoscreening and confirmed their authenticity by direct N-terminal protein sequencing. Although the deduced amino acid sequence of the 22-kDa subunit is not overtly similar to other known cytochromes, we observed a 31-amino acid stretch of 39% identity with polypeptide I of mitochondrial cytochrome c oxidase centered on a potential heme-coordinating histidine. Similarities in the hydropathy profiles and spacing of histidines of the 22-kDa protein and myoglobin suggest structural motifs in common with other heme-containing proteins that are not readily revealed by primary amino acid sequences. Although RNA for the larger subunit has been found only in cells of the phagocytic lineage, stable RNA encoding the 22-kDa subunit was observed in all cell types. However, the stable 22-kDa protein was detected only in phagocytic cells that were expressing the larger subunit RNA. This observation suggests that the large subunit may play a role in regulating the assembly of the heterodimeric cytochrome b.