Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC----AT transitions and 17% were AT----GC transitions. In addition, the number of AT----TA (20%) and AT----CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT----TA transversion mutations in the activation of cellular proto-oncogenes by ENU.     

DNA methylation in specific cells of rat liver by N-nitrosodimethylamine and N-nitrosomethylbenzylamine

Dose-response curves for the O6-methylation of guanine in the hepatic DNA of Wistar and Sprague-Dawley rats were determined after administration of N-nitrosomethylbenzylamine (NMBzA) or N-nitrosodimethylamine (NDMA). Similar results were obtained for both rat strains but methylation of hepatic DNA by NDMA was approximately 9-fold more efficient than with NMBzA when doses were compared on a molar basis. Comparison by immunohistochemical analysis of the distribution of nuclei containing O6-methylguanine within the liver lobules showed that both agents tended to alkylate cells close to the central veins at the lower doses. With increasing doses, the band width of alkylated cells around the central vein increased, spreading in the case of NDMA virtually into the portal zones, whereas with NMBzA the zone of alkylated nuclei reached little more than halfway from the central vein to the portal zone. These differences in the distribution of alkylated cells may explain the differing hepatic responses to these two nitrosamines.     

Rearrangements in minisatellite sequences induced by aflatoxin B1 in a metabolically competent strain of Saccharomyces cerevisiae

The role of aflatoxin B1 (AFB1) in the induction of rearrangements affecting minisatellite sequences was studied in an in vitro yeast model. The Saccharomyces cerevisiae strain used expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 oxidoreductase and has previously been used to study genetic recombination events induced by AFB1. DNA multilocus fingerprinting was performed using probe M13 core hybridizing to a set of hypervariable minisatellite sequences in S. cerevisiae. Frequent spontaneous genomic alterations that affect the minisatellite fingerprint pattern were observed. Control cultures showed 15.8% rearrangements in minisatellites, and this frequency increased to 40.0% in cultures exposed to AFB1 (80 microg/ml). A total of approximately 29 minisatellite loci were visualized for each culture. Given the number of cultures examined (40 AFB1-treated and 38 controls) the rearrangement frequency per detectable minisatellite was 2.59% in the AFB1-treated group and 0.73% in the control group, which represents a statistically significant (P = 0.001) difference. Thus, our data strongly suggest that AFB1 can promote the genetic events responsible for minisatellite rearrangements in the yeast genome. Such genetic rearrangements may be important events during the etiology of liver carcinogenesis in people chronically exposed to dietary aflatoxins.      

Analysis of mutations induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human lymphoblastoid cells

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclicaromatic amine that is formed in abundance in cooked meats,has been found to be mutagenic in human lymphoblastoid TK6 cellsat the thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase (hgprt) loci. The mutations induced at the hgprtlocus have been analysed. Of the mutations that have been identified,60% were found in the coding sequence of the gene. Forty percentwere in the introns which resulted in aberrant splicing andconsequently, leading to exon losses in the mature hprt mRNA.Mutations resulting in a loss of exonIII appeared most frequentlyfollowed by losses of exonVI, exonVIII and partial loss of exonIX.All identified mutations occurred at GC base pairs, consistentwith the adducts of PhIP that have been found previously andsuggesting that the N-(deoxyguanosin-8-yl)-2-amino-l-methyl-6-phenyl-imidazo[4,5-b]pyridine,(dG-C8-PhIP) adduct may be the premutagenic lesion. Most ofthe mutations are GC     

O6-Methylguanine removal by competent and incompetent human lymphoblastoid lines from the same male individual

Two cell lines, one proficient (Mex+) and one deficient (Mex-) in the ability to remove O6-methylguanine, have been isolated by Epstein-Barr virus-mediated transformation of a single blood sample obtained from a normal human male. Extracts of untreated cells differ in their O6-methylguanine transferase (methyl acceptor protein) activity. Although both lines arise from the same individual, they show great difference in their sensitivities to the toxic action of N-methyl-N'-nitro-N-nitrosoguanidine. Chromosome counts of the strains reveal a modal number of 46 for both. Neither X-inactivation nor a gross abnormality in chromosome number can be the cause of the difference between the two lines.     

Homozygous deficiency at autosomal locus aprt in human somatic cells in vivo induced by two different mechanisms

Associations between germinal and somatic mutations at autosomal loci play an important role in the development of some tumors, including retinoblastoma. In an attempt to determine whether equivalent events occur in vivo at other loci, we cloned and enumerated somatic T-cells with mutations at the aprt locus, by taking advantage of both the presence of a human disease caused by genetic defects at this locus and an effective selection procedure for the deficient mutants. T-cells homozygously deficient at this locus (aprt-/-) were found in all four heterozygotes (aprt+/-) studied, at an average frequency of 1.3 x 10(-4). From 310 normal individuals, we identified only one aprt-/- clone, and the calculated frequency of aprt-/- T-cells in aprt+/+ individuals was 5.0 x 10(-9). These results confirm that a two-step process (aprt(+/+)----aprt(+/-)----aprt-/-) is functional through two different mechanisms (germinal-somatic and somatic-somatic) in vivo. Our data suggest that the two-step mutations leading to homozygous deficiencies at the somatic cell level, as proposed for the carcinogenic mechanisms for retinoblastomas and other human tumors, generally occur at rather high frequencies at various autosomal loci in humans.     

A spectrum of mutations induced by crotonaldehyde in shuttle vector plasmids propagated in human cells

A spectrum of crotonaldehyde-induced mutations in the supF gene of the shuttle vector plasmid pMY189 replicated in human fibroblast cells was examined. Base sequence analysis of 104 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (85%) and the rest were frameshifts (15%). A single base substitution was most frequently found (47%), while 25% had multiple base substitutions and interestingly 13% had tandem (adjacent two) base substitutions. Of the base substitution mutations, 50% were G:C-->T:A transversions and 23% were G:C-->A:T transitions. The mutations were not distributed randomly but were located at several hotspots, most of which were G:C base pairs in 5'-AAGG-3' (or 5'-CCTT-3') sequences. Production of propanodeoxyguanosine adducts may be related to such specificity in the mutation spectrum.      

Induction kinetics of mutations at two genetic loci, DNA damage and repair in CHO cells after different exposure times to N-ethyl-N-nitrosourea

Ouabain resistance (oua^r) and 6-thioguanine resistance (6-TG^r)mutation frequencies were measured in Chinese hamster ovarycells after treatment with N-ethyl-N-nitrosourea (ENU) for varyingperiods of time. Maximal mutation frequency at the Na⁺/K⁺ ATPasegene locus (oua^r mutations) was attained within 5 min of exposure,whereas the mutation frequency at the hypoxanthine guanine phosphoribosyltransferaselocus (6-TG^r mutations) continued to increase up to 60 min,following the theoretical curve for exponential decay of ENUwith time. Detection of DNA single strand breaks (ssb) by alkalineelution showed that maximal levels were at tained within 5 minof treatment with ENU. Fast repair of DNA ssb occurred earlyafter exposure (>50% repair within 10 min). Analysis of DNAethylation products by h.p.l.c. showed initially rapid removalof O²-ethylcytosine (25% in the first hour), slow removal of7-ethylguanine, 3-ethyladenine and 3-ethylguanine and no removalat all of O⁶-ethylguanine, O⁴-ethylthymine and ethylphosphotriesters.These time-course studies reveal different target gene responsesin the fixation of DNA damage into mutations.     

Quantitative analysis of cytotoxicity induced by HTLV-I carrying cells against a human lymphoblastoid cell line, Molt-4

Co-cultivation of HTLV-I carrying cells and virus-free lymphoid cells resulted in the cytopathic effect and cytotoxicity of the latter cells. Microscopically, these phenomena were observed as early as 2 h as the appearance of multinuclear giant cells and ballooning cells with striking resemblance with cytopathic changes induced by HTLV-III. When the cytopathic effect and cytotoxicity of the target cells by HTLV-I carrying cells were assayed by 51Cr-release assay system, these phenomena were quantitatively analysed and the cytotoxic activity was observed generally in HTLV-I positive cells. Virally induced cytotoxicity was inhibited by plasma of adult T-cell leukemia (ATL) patients specifically. Cytotoxic activity of HTLV-I positive cell lines was correlated with HTLV-I antigen expression of them. HTLV-I negative cell lines did not express cytotoxicity significantly.     

Aldehyde dehydrogenase-1 is a specific marker for stem cells in human lung adenocarcinoma

To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation, colony formation, and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (“pulmospheres”) were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability, high cloning efficiency, and strong tumorigenicity. The rate of SSC^loALDE^br cell colony formation was 1.3–5.6%, whereas that of ALDE⁻ cell colony formation was only 0–1.2% (P < 0.05). A cell count of only 1 × 10³ SSC^loALDE^br cells was necessary to form tumors, whereas at least l × 10⁵ ALDE⁻ cells formed tumors. The same number of SSC^loALDE^br cells also formed larger tumors in a short latency period of tumor formation. The expression rates of CD133 in the SSC^loALDE^br and ALDE⁻ cells were 16.31% (16.31 × 10⁴/10⁶) and 2.56% (2.56 × 10⁴/10⁶), respectively (P < 0.01). Moreover, the expression rates of ABCG2 in SSC^loALDE^br and ALDE⁻ cells were 17.62% (17.62 × 10⁴/10⁶) and 3.45% (3.45 × 10⁴/10⁶), respectively (P < 0.01). Human lung adenocarcinoma bears CSCs, and ALDH-1 can act as a specific marker for human lung CSCs.     

Estrogen treatment induces MLL aberrations in human lymphoblastoid cells

Epidemiological data indicates increased risk of infant acute leukemia involving MLL gene aberrations with use of oral contraceptives. To determine whether estrogens might be implicated, we examined the effect of estradiol (E2) or 4-OH-E2 in an in vitro model of translocation susceptibility. Genomic DNA from the TK6 human lymphoblastoid cell line was screened by ligation mediated PCR and inverse PCR at a rearrangement hot spot within the MLL breakpoint cluster region to detect DNA aberrations. An increase in DNA double strand breaks was observed within this region after exposure to either E2 or 4-OH-E2. An increase in the frequency of MLL translocations was only found after exposure to E2. Induction of cleavage due to increased activation of apoptotic nucleases was excluded by pre-treatment with the pan-caspase inhibitor, zVAD.fmk. We conclude that concentrations of E2 and 4-OH-E2 that may occur during pregnancy, or during use of oral contraceptives, can cause aberrations of the MLL gene and could thus be a factor in the early events of leukemogenesis occurring in utero.     

Epstein-barr virus-induced cap formation in human lymphoblastoid cells.

Redistribution and consequent cap formation of Epstein-Barr virus adsorbed to human lymphoblastoid cells were studied by indirect membrane immunofluorescence carried out at 0 degrees C. When EBV was adsorbed on cells at 0 degrees C, the cell surface fluorescence had a mostly r-ng-like pattern. However, the ring cells could be transformed into cap cells when warmed at 37 degrees C. This cap formation could be induced by EBV alone without participation of antibodies involved in the immunofluorescence procedure. The cap formation was temperature- and pH-dependent, and was reversibly inhibited by sodium azide or some sugars. Thus the EBV-induced cap formation was analogous to that induced by antibodies or ligands on other lymphoid cells.     

Induction of sister chromatid exchange by diethylstilbestrol in metabolically competent hepatoma cell lines but not in fibroblasts

The effect of diethylstilbestrol (DES) on sister chromatid exchange (SCE) induction was measured in four cell lines to determine whether metabolic activation of DES is a factor in its genotoxic potential. Two of these, cell lines derived from a human hepatoblastoma (HepG2-GW) and a rat hepatoma (H4-AG), have been shown previously in our laboratory to be capable of metabolizing several procarcinogens to their active forms. DES, in a dose range of 1 X 10(-8) M to 1 X 10(-5) M, increased SCE frequencies by 50 to 60% in both the rat and human hepatoma lines but had no effect on SCE induction in Chinese hamster lung fibroblasts (V79-GW) or human diploid skin fibroblasts (MGH 2C-GW), both of which are nonmetabolizing cell lines. Furthermore, pretreatment of the responsive cell lines (H4-AG and HepG2-GW) with indomethacin, an inhibitor of prostaglandin synthetase-mediated metabolism of DES, effectively prevented the induction of SCE by DES. DES failed to increase the frequency of hypoxanthine-guanine phosphoribosyltransferase locus mutants in H4-AG cells, over a dose range which induced SCE. These observations suggest that DES induces SCE but does not induce gene mutation. These data strongly support growing evidence that metabolic activation of DES may be an important factor in its genotoxic and carcinogenic mechanisms.     

Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells.

We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis. A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.     

Frequent and multiple mutations at minisatellite loci in sporadic human colorectal and gastric cancers--possible mechanistic differences from microsatellite instability in cancer cells.

Minisatellites (MNs), composed of 5 to 100 nucleotide repeat units, range from 0.5 to 30 kb in length, and have been reported to be mutated in various human malignancies. In this study, frequencies of MN mutations in sporadic human colorectal (34 cases) and gastric cancers (24 cases) at various clinicopathological stages were assessed by multilocus DNA fingerprint analysis with three MN probes, Pc-1, 33.6 and 33.15. MN mutations were observed in both colorectal and gastric cancers, but at a significantly higher frequency in the former (56%) than in the latter (25%). Multiplicities of MN mutations were 1.50 +/- 1.81 and 0.46 +/- 1.10 in colorectal and gastric cancers, respectively, and the difference was also significant. Neither the presence nor multiplicity of MN mutations in either colorectal or gastric cancer cases had any correlation with the pathological stage, histological grading or the presence of microsatellite instability (MSI). Although the biological relevance of MN mutations still remains to be clarified, a subset of colorectal and gastric cancers could feature a new type of genomic instability, distinct from MSI.     

Elaboration of pyrimidine-specific nucleosidases by human lymphoblastoid cells of established cultures.

Pyrimidine-specific nucleosidases were released rapidly by human lymphoblastoid cells of established cultures when incubated under certain culture conditions having no adverse affect on their viability or morphology. Nucleosidase production was not restricted to any particular type of lymphoblastoid line; enzymes with a high level of activity were elaborated by cells of cultures initiated from healthy subjects and patients with uncontrolled lymphocytic or myelocytic leukemia, as well as by cells of cultures exhibiting mostly B- or T-cell properties. Tritiated thymine and uracil, which were not incorporated to any appreciable extent by DNA- and RNA-synthesizing cells, were identified by paper chromatography as the primary products arising from nucleosidase degradation of radiolabeled thymidine, uridine, and cytidine. Neither adenosine nor guanosine was catabolized. These heat-labile and ultraviolet-sensitive enzymes with a molecular weight of 5 to 10 X 10(4) did not affect the viability, morphology, or proliferation of lymphocytes in mitogenactivated cultures, lymphoblastoid cells in long-term cultures, or fibroblasts in monolayer cultures.     

HeLa细胞HSP-70体外诱导抗瘤免疫活性细胞的研究

背景与目的:近来的研究证实,动物肿瘤细胞表达的热休克蛋白(heatshokproteinHSP)可携带肿瘤抗原,并能诱导抗瘤特异性T淋巴细胞的免疫应答。本文旨在探讨用HeLa细胞HSP-70多肽诱导的免疫活性细胞(immunecompetentcell,ICC)及其特异性抗肿瘤机制。方法:用热休克处理的HeLa细胞提取的HSP-70多肽剌激正常人外周血单个核细胞(PBMCs),在剌激扩增前后测定T淋巴细胞表型,并测定扩增的免疫活性细胞对HeLa细胞的杀瘤活性。结果:在扩增前后CD3+淋巴细胞百分率为(57.68±1.46)%和(85.73±1.44)%(P<0.002),CD8+淋巴细胞百分率为(23.56±1.86)%和(48.06±1.66)%(P<0.002)。该免疫活性细胞对HeLa细胞和来源于病人的宫颈癌细胞都具有较高的杀伤活性犤(82.69±1.97)%和(62.11±1.61)%犦,而对经HSP-70单抗封闭后的HeLa细胞的杀伤活性犤(31.05±2.09)%犦则明显下降。结论:HeLa细胞HSP-70多肽能有效刺激PBMCs诱导活化免疫活性细胞,该免疫活性细胞具有特异性抗肿瘤作用。