Search PubMed⌕ Search

Biomedical subjects

J J McCormick

Publications and source records attributed to J J McCormick.

At least 73 records · Page 4Linked to original sources
PubMed1990

Malignant transformation of human fibroblasts by oncogene transfection or carcinogen treatment.

Exposure to chemical carcinogens or radiation is considered to cause most human cancer, but human cells in culture have not been successfully transformed to malignancy by such agents. Malignant transformation is a multi-step process and one explanation for the failure to induce such transformation of human cells in culture could be inability to recognize the phenotypes of carcinogen-treated cells that have undergone intermediate changes, so that these cells can be isolated and exposed a second time to cause further changes. To identify possible intermediates, we transfected diploid human fibroblasts with oncogenes known to be active in cells derived from fibrosarcomas and determined the phenotypes produced. H- or N-ras oncogenes flanked by suitable enhancer and promoter sequences caused the cells to exhibit several characteristics of malignant cells, but not to acquire an infinite life span or form tumors. Transfection of these oncogenes in the same constructions, or a viral K-ras oncogene, into an infinite life span, near-diploid, non-tumorigenic cell strain developed in this laboratory (MSU-1.1 cells) resulted in distinct foci of morphologically-altered, anchorage independent, and growth factor independent cells that formed progressively-growing, invasive malignant sarcomas in athymic mice and expressed the p21s of the transfected ras genes. Transfection of two other infinite life span human cell lines with the H-ras oncogene in the same construction also yielded malignant cells. Recently, we succeeded in inducing the malignant state in MSU-1.1 cells using carcinogen identified that are one step removed from malignant transformation, others that are two-steps removed, etc. Furthermore, we know what new phenotypes these cells need to express to be malignantly transformed and which oncogenes can make such a change. If, as suggested above, proto-oncogenes are the cellular targets for carcinogen attack, it should be possible, by carcinogen treatment to bring about the malignant state. We have recently succeeded in achieving just such transformation by exposing MSU-1.1 cells to chemical carcinogens.

Animals↗
PubMed1990

Malignant transformation of an infinite life span human fibroblast cell strain by transfection with v-Ki-ras.

To determine if human fibroblasts can be transformed into malignant cells by transfection of a K-ras oncogene, we transfected the provirus of Kirsten murine sarcoma virus (v-Ki-ras) into an infinite life span human cell strain, MSU-1.1, which has a normal morphology, is not anchorage independent, and has a stable, near-diploid karyotype. The transfected populations gave rise to distinct foci composed of morphologically-altered cells. The cells from several independent foci were isolated, propagated, and assayed for anchorage independence and/or tumorigenicity. They formed large-sized colonies in soft agar at a high frequency. Cell strains derived from colonies isolated from agar as well as focus-derived cell strains were injected subcutaneously into athymic mice to test for tumorigenicity. One cell strain yielded myxoid fibromas, the rest produced well-differentiated, progressively-growing, invasive, myxoid or spindle cell sarcomas. The karyotype of each of the cell strains tested, including cell strains derived from tumors, was identical to that of non-transfected MSU-1.1 cells. Two focus-derived strains, and two cell strains derived from sarcomas produced from them, were tested and shown by DNA and RNA hybridization to contain and express the v-Ki-ras oncogene. Radioimmunoprecipitation analysis showed that these strains expressed ras-specific p21 products not found in non-transfected MSU.1.1 cells. When injected intraperitoneally, a cell strain derived from a myxoid tumor gave rise to invasive myxoid tumors at various sites in the body. The same cell strain gave rise to invasive spindle cell sarcomas when injected into the tail vein of the animals.

Blotting, Northern↗
PubMed1989

Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells.

The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.

Base Sequence↗
PubMed1989

Malignant transformation of mammalian cells in culture, including human cells.

This overview of the malignant transformation of mammalian cells in culture, including human cells, describes the earliest evidence of spontaneous, virus-induced, and carcinogen-induced transformation. It discusses several systems developed to assay the carcinogen-induced transformation of highly selected infinite life span ("established") cell lines as well as finite life span diploid cells. Evidence is presented to support the multistep hypothesis of the process of malignant transformation, and the theoretical requirement for acquisition of an infinite, or greatly extended, life span in culture if a cell is to become malignant is explained in light of the multistep nature of the process. The use of oncogene transfection studies to analyze the number and kinds of changes involved is discussed, with emphasis on studies using human cells. Finally, the results of earlier studies on viral- and carcinogen-induced transformation of mammalian cells (or chicken cells) are reinterpreted in the light of more recent insights into the process of carcinogenesis.

Animals↗
PubMed1989

Effect of retinoids on growth factor-induced anchorage independent growth of human fibroblasts.

We have studied the effects of all-trans retinol, all-trans retinoic acid, and anhydroretinol, a biologically inactive retinoid, on anchorage-independent growth of human fibroblasts induced by purified growth factors. The anchorage-independence assay was conducted in medium supplemented with serum that had had its peptide growth factors inactivated by treatment with dithiothreitol and iodoacetamide. Physiologic concentrations of either all-trans retinol (0.5 microM) or all-trans retinoic acid (1.0 nM) but not anhydroretinol (0.5 microM) reduced the frequency of anchorage-independent growth of normal human fibroblasts induced by platelet-derived growth factor (PDGF). All-trans retinol was also tested for its effect on the frequency of anchorage-independent growth induced by basic fibroblast growth factor (bFGF) and was found to decrease this growth. All-trans retinol also reduced the frequency of anchorage-independent growth of the human fibrosarcoma-derived cell line, HT1080, which grew in semisolid medium without added growth factors. Inasmuch as these retinoids reduced the frequency of anchorage-independent growth induced by either PDGF or bFGF and because PDGF and bFGF bind to independent cell membrane receptors and are known to stimulate different pathways leading to DNA synthesis, the data suggest that physiologically active retinoids have an effect on a step that is common to both signal pathways.

Cell Adhesion↗
PubMed1989

Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

To investigate the role of DNA damage in the induction of homologous recombination in mammalian cells, a series of structurally related, polycyclic aromatic carcinogens, i.e., 1-nitrosopyrene (1-NOP), N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and 4-nitroquinoline 1-oxide (4-NQO), were compared for their ability to cause intrachromosomal homologous recombination between two herpes simplex virus thymidine kinase (Htk) genes stably integrated in the genome of a tk- mouse L cell strain 333 M. Each Htk gene contains an 8-bp XhoI linker inserted at a unique site so that expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. Each carcinogen caused a dose-dependent increase in the frequency of recombination. The results were compared to what had been found previously for a structurally related carcinogen, (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). As a function of concentration, BPDE was the most active agent, followed by 4-NQO, and 1-NOP, and then N-AcO-AAF. When compared on the basis of equal cell killing, the most efficient carcinogen was 1-NOP, followed by N-AcO-AAF and BPDE, and then 4-NQO. Use of tritium-labeled compounds to determine the frequency of recombination as a function of the number of adducts initially bound to DNA showed that the most effective agent was BPDE, followed by 1-NOP and 4-NQO, and then N-AcO-AAF (ratio, 6.6:2.5:1.8:1.0). To determine if these differences in recombinagenic effectiveness reflected different rates of removal of the adducts from DNA, we measured the percentage of DNA adducts removed during the 24-h period post treatment and found that 1-NOP, 4-NQO and N-AcO-AAF residues were removed at approximately the same rate, i.e., 25%-30% off. Cellular analysis of a series of independent recombinants indicated that approximately 82% of the recombinational events induced by each agent were consistent with gene conversion. DNA-DNA hybridization analysis confirmed this, and showed that each recombinant tested contained an XhoI-resistant (wild-type) Htk gene; with the majority retaining the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids.

4-Nitroquinoline-1-oxide↗
PubMed1989

Malignant transformation of human fibroblasts caused by expression of a transfected T24 HRAS oncogene.

We showed previously that diploid human fibroblasts that express a transfected HRAS oncogene from the human bladder carcinoma cell line T24 exhibit several characteristics of transformed cells but do not acquire an infinite life-span and are not tumorigenic. To extend these studies of the T24 HRAS in human cells, we have utilized an infinite life-span, but otherwise phenotypically normal, human fibroblast cell strain, MSU-1.1, developed in this laboratory after transfection of diploid fibroblasts with a viral v-myc oncogene. Transfection of MSU-1.1 cells with the T24 HRAS flanked by two transcriptional enhancer elements (pHO6T1) yielded foci of morphologically transformed cells. No such transformation occurred if the plasmid containing T24 HRAS had only one enhancer or none at all or if the normal human HRAS gene was transfected in the pHO6 vector (pHO6N1). Cell strains derived from such foci expressed high levels of T24 HRAS product p21, formed colonies in soft agar at high frequency, proliferated rapidly in serum-free medium that does not support growth of the parental cell line, and formed progressively growing, invasive fibrosarcomas. These foci-derived T24 HRAS-transformed cell strains, as well as cells from the tumors derived from them, had the same near-diploid karyotype as that of the parental MSU-1.1 cells. Transfection of pHO6T1 into two other infinite life-span human fibroblast cell lines, cells that had not been transfected with v-myc, also resulted in malignant transformation, suggesting that the infinite life-span phenotype of MSU-1.1 cells, and not necessarily expression of the v-myc oncogene, was the factor that complemented T24 HRAS expression to cause malignant transformation.

Cell Line↗
PubMed1989

Mutations induced by aminofluorene-DNA adducts during replication in human cells.

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we treated a shuttle vector, pZ189, carrying the supF gene as the target for mutations with N-acetoxy-N-trifluoroacetyl-2-aminofluorene (N-AcO-TFA-AF). The plasmids were allowed to replicate in human cell line 293, and the progeny plasmids were examined for the frequency and kinds of mutations induced in supF, as well as their specific location in the sequence of the supF gene. The plasmids were reacted with N-AcO-TFA-AF so as to obtain the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), the principal adduct formed in DNA when mammalian cells are exposed to reactive derivatives of 2-acetylaminofluorene (AAF), including N-acetoxy-2-acetylaminofluorene. The results showed there was a linear relationship between the number of dG-C8-AF adducts per plasmid and the frequency of supF mutants induced. DNA sequencing of 47 independent mutants obtained from doses of N-AcO-TFA-AF that increased the frequency of mutants 9-15 times the background frequency and three independent mutants from lower doses showed that 92% contained point mutations, i.e. changes affecting one, or two, or three nearby bases, and that all of these point mutations involved G.C base pairs. Ninety eight percent of the point mutations were base substitutions, predominantly G.C----T.A transversions. 46% of these mutations occurred at four out of the 85 bp in the target gene (hot spots). The most prominent mutation hot spot was also the most prominent hot spot for adduct formation as judged by the frequency of termination of in vitro polymerization by the Klenow fragment on N-AcO-TFA-AF-treated plasmids.

2-Acetylaminofluorene↗
PubMed1989

Transformation of diploid human fibroblasts by transfection of N-ras-oncogenes.

To determine if diploid human fibroblasts can be transformed by the N-ras oncogene found in human tumors, early passage cell lines were transfected with an N-ras oncogene from human leukemia cell line 8402 cloned into a high expression plasmid (pSV N-ras), with the N-ras oncogene from human fibrosarcoma cell line HT1080 cloned into pNR-MG1, or with pSV2neo as a control. Each plasmid carries the neo gene coding for Geneticin resistance, but in pSV N-ras, the endogenous promoter of the N-ras gene has been eliminated, and the gene has been inserted between the viral long-terminal repeat (LTR) and the neo gene so that transcription initiated from the LTR must transcribe the N-ras gene before transcribing neo. In pNR-MG1, transcription of the N-ras gene is driven from its endogenous promoter, and the neo gene is transcribed from an SV40 viral promoter, as in pSV2neo. When the transfectants were selected for Geneticin resistance, 70% of the colonies formed with pSV N-ras consisted of morphologically altered cells. Less than 5% of the drug resistant colonies formed with pNR-MG1 had cells with altered morphology, and the change in morphology was much less distinct than with pSV N-ras. No colonies of morphologically-transformed cells were found with pSVneo. If the transfected populations were not selected in Geneticin, but were simply allowed to grow to confluence, very distinct foci composed of morphologically-altered cells could be seen against a contact-inhibited monolayer with pSV N-ras. Foci formed by the pNR-MG1 population were subtle and much less distinct. No foci were found with pSV2neo. Representative colonies and foci of morphologically-transformed cells were isolated. Those from pNR-MG1 transfection reverted to a normal morphology after 5-10 population doublings. Most of those from pSV N-ras transfection either reverted or senesced after 5-10 population doublings. However, progeny of two colonies expressed stable morphological transformation throughout a normal life span equal to that of age-matched pSV2neo controls. Both of these stably transformed cell strains exhibited anchorage independence and formed distinct foci. Immunoprecipitation analysis showed that these cells produced much larger amounts of N-ras protein than did pSV2neo-transfected control cells. However, these two cell strains did not exhibit an infinite life span in culture and were unable to form tumors in athymic mice.

Cell Line↗
PubMed1988

Relationship between sensitivity of cells from patients with hereditary cutaneous malignant melanoma to killing and mutations by 4-nitroquinoline 1-oxide and adduct formation.

Hereditary cutaneous malignant melanoma (HCMM) is an autosomal dominant disease and develops mostly in patients with the hereditary dysplastic nevus syndrome (DNS). A previous study from this laboratory showed that fibroblasts derived from skin biopsies of four HCMM/DNS patients and one DNS patient were significantly more sensitive to the cytotoxic and mutagenic effects of 4-nitroquinoline 1-oxide (4-NQO) than three fibroblast cell lines from normal newborn males and one from a 45-yr-old normal donor. The survival curves of the HCMM/DNS or DNS cell lines fell into two groups, one with a slope approximately twice as steep as the controls and one with a slope approximately 3 times as steep. This marked difference in survival curves disappeared if the same cell lines were exposed to 4-hydroxyaminoquinoline 1-oxide, a partially reduced metabolite of the parent compound, suggesting that the difference in sensitivity reflected a difference in the cells' ability to metabolize the parent compound into a more reactive form. To see if such increased sensitivity to the cytotoxic effect of 4-NQO was an identifying characteristic of cells from HCMM/DNS and DNS patients and whether cells derived from unaffected members of such families were also more sensitive to 4-NQO than the controls, the survival curves of a series of 40 coded cell lines, derived from patients, family members, or normal donors of various ages, were compared in a blind study. The slopes of the survival curves of all the HCMM/DNS and DNS cell lines proved to be 2- to 3-fold steeper than those of the four previously studied control cell lines, bringing the total to nine of nine and eight of eight, respectively. Ten of the 28 coded cell lines from nonaffected family members or normal, age-matched donors proved to be as resistant as the four previously studied control cell lines and another foreskin-derived control cell line, bringing the total of relatively resistant cell lines to 15 of 33. However, the rest were just as sensitive as the cells from the patients. Mutagenesis studies showed that increased sensitivity to killing by 4-NQO was accompanied by increased sensitivity to mutation induction. Relatively sensitive and relatively resistant cell lines were treated with tritiated 4-NQO and assayed for survival and for the number of 4-NQO residues bound to DNA. The relationship between the number of adducts formed and cell survival was linear, with the relatively sensitive cell lines exhibiting the higher level of residues.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitroquinoline-1-oxide↗
PubMed1988

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts.

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.

Blood↗
PubMed1988

Towards an understanding of the malignant transformation of diploid human fibroblasts.

This paper reviews the major reports of the spontaneous or carcinogen-induced transformation of human fibroblasts to the malignant state, to infinite lifespan, or to anchorage independence. In some cases, the transformed cells and the parent cell with which the work began were made available to us to be tested to determine whether the cells shared common isozymes, HLA antigens, restriction-fragment length polymorphisms, marker chromosomes, etc., as one would expect. When we examined the normal fibroblastic cell line KD for these markers, and the transformed HuT cell lines developed from it by Kakunaga (Proc. Natl. Acad. Sci. (U.S.A.), 75, 1334, 1978) for these markers, we found marked differences, indicating that KD cells and HuT cells are derived from different individuals. When we applied these techniques to the 3 human fibroblast cell lines transformed by Namba to acquire infinite lifespan in culture (Gann, 27, 221, 1981), it became clear that KSMT-6 was derived from the parent cell, KMS-6, but that both cell lines CT-1 and SUSM-1 were derived from the same parental cell line, AD387. Similar studies with other sets of cell lines are also reported. In the light of these studies, it appears that there is no example of the malignant transformation of human fibroblasts by carcinogen treatment. However, neoplastic transformation and transformation to infinite lifespan by carcinogen treatment have been achieved by a number of workers. We speculate as to how malignant transformation might be obtained by carcinogen treatment.

Carcinogens↗
PubMed1988

Transformation of diploid human fibroblasts by transfection with the v-sis, PDGF2/c-sis, or T24 H-ras genes.

Gene transfection techniques have provided powerful methods to examine the roles of cellular and retroviral oncogenes in the transformation process in rodent fibroblasts. However, the use of such techniques with diploid human fibroblasts has been limited. We have developed transfection procedures to reproducibly transfect such cells with oncogenes, and methods for the biological characterization of the transformants. We have shown that the v-sis and T24 H-ras oncogenes, as well as the platelet-derived growth factor gene (PDGF2/c-sis), are capable of inducing a transformed phenotype in normal diploid human fibroblasts, but are not capable of conferring infinite lifespan or making such cells tumorigenic.

Cell Transformation, Neoplastic↗
PubMed1988

The HuT series of 'carcinogen-transformed' human fibroblast cell lines are derived from the human fibrosarcoma cell line 8387.

In 1977 Kakunaga reported the carcinogen-induced transformation of the diploid human fibroblast cell line KD into focus-forming, morphologically altered cells. Cell lines were developed from 15 individual foci. These exhibited an infinite lifespan in culture and all those that were tested (7/7) formed malignant tumors (sarcomas) in athymic mice. The existing cell lines, designated HuT-11 to HuT-14, have been studied intensively during the past decade as examples of human fibroblasts malignantly transformed by treatment with a chemical carcinogen, 4-nitroquinoline-1-oxide. Recently, in comparing the HuT-11, HuT-12 and HuT-14 cell lines with KD cells, McCormick and Maher (Mutat. Res., 199, 273-291, 1988) found evidence that the malignant cells could not have been derived from the latter. But, this did not rule out the possibility that as the target cells for his original study of carcinogen-induced transformation, Kakunaga had inadvertently used cells from some other, unidentified normal individual. Since the donor of such cells would not be known and the original cell line was not available, it would be impossible to determine the degree of identity between such a target cell line and the HuT cell lines. However, in the course of examining methods for such testing, we recently became aware that the isozyme pattern of these HuT cell lines was identical to that of the human fibrosarcoma-derived cell line 8387 established in 1966. We here report that the HuT cell lines and the 8387 cell line also exhibit an identical series of HLA determinants and identical restriction fragment length polymorphisms (RFLPs). Assuming that each of these three assays measures independently inherited characteristics, the chance that an unrelated donor of the fibroblasts that gave rise to the HuT cell lines happened to possess characteristics identical to those of the patient whose fibrosarcoma gave rise to the 8387 cell line is 1 x 10(-8). Therefore, we conclude that 8387 cells are the source of the malignant cells designated HuT from Kakunaga's original transformation experiment. Additional RFLP analysis, using a probe made from M13 bacteriophage DNA which detects a hyperpolymorphic 'minisatellite' pattern in human DNA, also showed that DNA from HuT-14 cells and from 8387 cells exhibit identical banding patterns, indicating that the cell lines were taken from the same individual. The latter banding patterns differed from that observed with DNA from KD cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.

Base Sequence↗
PubMed1988

Studies on the metabolism and biological effects of nitropyrene and related nitro-polycyclic aromatic compounds in diploid human fibroblasts.

Nitro derivatives of polycyclic aromatic hydrocarbons are produced primarily as the result of incomplete combustion. Nitropyrenes have been identified as primary mutagenic compounds of diesel emission particulate and are tumorigenic in laboratory animals. Since nitropyrenes do not react directly with DNA, their effects presumably are mediated through cellular conversion of the parent compounds into reactive species. For example, 1-nitropyrene (1-NP) is activated by enzymatic reduction to 1-nitrosopyrene (1-NOP), followed by reduction to the hydroxylamine, which undergoes decomposition to yield a nitrenium ion, that reacts with DNA. The cytotoxic effects of 1-nitropyrene and 1-nitrosopyrene were compared in fibroblasts from normal persons, from excision-repair-deficient xeroderma pigmentosum (XP) patients, and from a patient with an inherited predisposition to malignant melanoma of the skin (hereditary cutaneous malignant melanoma [HCMM]). HCMM cells are more sensitive than normal cells to the cytotoxic and mutagenic effects of 4-nitroquinoline-1-oxide, and they form more DNA adducts per concentration of this agent than do normal cells. However, the HCMM cells exhibit the same sensitivity as normal cells to 4-hydroxyaminoquinoline-1-oxide, which suggests they are more capable than normal cells of metabolizing the parent compound into a more reactive form. On the basis of concentration, 1-NOP was much more cytotoxic than 1-NP. With both compounds, the normal cells exhibited a shoulder on their survival curves that was lacking for the XP cells. The dose of 1-NP giving 37% survival was 46 microM for a series of four normal cell lines, 22 microM for the HCMM cell line tested, and 12 microM for the XP cell line. The slope of the 1-nitropyrene survival curve for XP cells was 2.5 times steeper than the slope of the curve of the normal cells; the slope of the 1-NP survival curve for the HCMM cells was intermediate between the XP cells and the normal fibroblasts. The slope of the 1-nitrosopyrene survival curve for XP cells was also 2.5 times steeper than that for the normal cells, but the HCMM cells showed a normal response. If the resistance of normal cells to the cytotoxic effect of these compounds reflects their ability to remove potentially cytotoxic adducts from their DNA before these lesions cause cell death, normal cells should require a higher initial number of DNA adducts than XP cells do to cause a particular degree of cell killing.(ABSTRACT TRUNCATED AT 400 WORDS)

Carcinogenicity Tests↗