Differential conservation of non-coding regions within human and guinea pig N-ras genes.
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Amino Acid Sequence↗
Biomedical subjects
J Doniger
Publications and source records attributed to J Doniger.
Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells.
A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangements. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.
Animals↗
In vitro models for studying the molecular biology of carcinogenesis.
Although carcinogens cause various similar deleterious effects on rodent and human cells, only rodent cells can convert to malignancy in a quantitative, predictable fashion. Therefore, the control mechanisms involving indefinite proliferation and tumorigenicity are different. Human cell lines may exhibit normal or aneuploid chromosome constitutions with numerical or structural alterations frequently involving proto-oncogene loci, but fail to produce progressively growing tumors in nude mice. A new approach for obtaining human cells susceptible to malignant transformation by chemical or physical carcinogens is to use DNA from a cancer associated virus. Transfection of human papilloma virus (HPV) DNA associated with genital cancer can extend life-span of human cells; post-X-irradiated cells grow in agar suspension. Southern blot analysis of extracted DNA indicates that HPV sequences persist. Similar results are obtained with human fibroblast and epithelial cells.
Animals↗
Altered polypeptide expression associated with neoplastic transformation of Syrian hamster cells by bisulfite.
Neoplastic transformation of Syrian hamster fetal cells by bisulfite is associated with qualitative and quantitative polypeptide changes. Amino acid-labeled [14C]polypeptides from neoplastic and nontransformed parental fetal cells were separated by two-dimensional gel electrophoresis and analyzed by computerized microdensitometry of autoradiographic patterns. Approximately 1000 polypeptides from parental fibroblasts at population doublings ranging from 4 to 20, and those from colony-derived malignant cell lines were compared. Most were identical. Seven malignant lines exhibited 4 qualitative polypeptide changes: 2 polypeptides had shifted slightly to the acidic side, 1 new polypeptide was observed, and 1 polypeptide was absent. The transformed bisulfite lines differed quantitatively from control cells in that 10-25% and 2-4% of the polypeptides exhibited differences in expression greater than 2- and 4-fold, respectively. Furthermore, there were 21 specific polypeptides with coordinate quantitative changes in all transformed lines. Because bisulfite at neutral pH fails to induce any significant DNA changes at concentrations that cause transformation, polypeptides expressed immediately or 48 h after bisulfite treatment were compared to those of non-treated controls, and no differences were found. Even though bisulfite does not induce detectable DNA damage or early post-treatment changes in polypeptide expression, a consistent set of qualitative and quantitative changes were observed after transformation. The qualitative polypeptide changes found in the bisulfite-induced malignant lines were similar to those seen in a benzo(a)pyrene-induced malignant line. This suggests that there is a convergence of pathways responsible for carcinogenesis independent of the nature of initiation.
Animals↗
Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.
Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.
Animals↗
Enhancement and inhibition of transformation of Syrian hamster embryo cells.
Diploid Syrian hamster embryo cells are particularly appropriate for the study of the transformation phenomenon in target cells. In vitro morphologic transformation occurs in a dose-dependent manner and is characterized by random crisscrossing and piling of cells; it correlates with tumorigenicity because individually transformed cell colonies can be isolated, cell lines can be developed, and the formation of tumors can be demonstrated after the injection of the transformed cells into either Syrian hamsters or athymic nude mice. HEC can also be used to investigate stages of carcinogenesis, initiation, and promotion. The susceptibility of normal HEC to transformation by environmental carcinogens including asbestos, bisulfite, nitrated non-carcinogenic polycyclic hydrocarbons, and X- or ultraviolet irradiation has made possible the determination of a variety of cell responses as they proceed to the neoplastic state. The initiation is usually a hereditary process involving single-hit kinetics and the transformation data indicate there is no measurable threshold response to carcinogens. The promotional aspects of transformation are readily modulated by environmental factors and have a threshold, as well as a maximal effect. The results of transformation studies using hamster cells indicate that in vitro studies are relevant to carcinogenesis and indicate that the various steps involved can be identified. Therefore, it should be possible to intervene with the various stages or steps leading to neoplasia so that cancer can be prevented.
Animals↗
Molecular characterization of oncogenes in guinea pig lines chemically initiated in vitro: acquisition of tumorigenicity is associated with activated ras related oncogenes.
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Animals↗
A transforming ras gene in tumorigenic guinea pig cell lines initiated by diverse chemical carcinogens.
Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.
Animals↗
Modulation of radiation induced transformation by combinations of a phorbol diester and a lymphotoxin.
The susceptibility of normal Syrian hamster embryo cells to transformation by environmental carcinogens has made possible the determination of a variety of responses as cells proceed to the neoplastic state. Expression of the initiated and promoted stages of irradiation carcinogenesis, for example, can be modified by cell surface alterations. Phytohemagglutin (PHA) or its isolectins decrease 12-O-tetradecanolphorbal-13-acetate (TPA) promoted transformation whereas PHA does not affect carcinogen only induced transformation. In contrast, both initiated and promoted transformation are sensitive to hamster lymphotoxin, a hormone-like, non-antibody lymphocyte glycoprotein. A 48 hr lymphotoxin treatment before or immediately after X-irradiation, or during TPA exposure causes a persistent inhibition independent of when carcinogen was added. A 6 hr lymphotoxin pulse before irradiation and TPA causes a persistent but nonpermanent effect unless followed by the carcinogen treatment; lymphotoxin becomes more potent as the interval between the lymphotoxin pulse and carcinogen insult or TPA addition is reduced. PHA and lymphotoxin affect the biological activity of TPA by diverse mechanisms. PHA may alter either the binding of TPA to a critical cellular receptor for promotion or alter a later step in promotion. Lymphotoxin can prevent the initiation of transformation and modulate carcinogenesis as well at both initiated and promoted stages in the transition to the neoplastic state.
Animals↗
Inhibition of radiation-initiated and -promoted transformation of Syrian hamster embryo cells by lymphotoxin.
Sensitivity of Syrian hamster cells to the anticarcinogenic action of hamster lymphotoxin obtained from mitogen-stimulated peritoneal lymphocytes depends on the stage of transformation, initiation, and promotion. Dose-response results with 12-O-tetradecanoylphorbol-13-acetate (TPA) plus X-irradiation parallel those obtained on mouse skin. Twice as much lymphotoxin was required to obtain a 50% reduction in TPA-promoted transformation as in nonpromoted transformation, demonstrating a difference in initiated and promoted cell sensitivity to lymphotoxin. In a study of promoted transformation, 48-hr lymphotoxin treatment before or immediately after X-irradiation, or during TPA exposure, caused a persistent inhibition independent of when lymphotoxin was added. The degree of sensitivity of different steps in carcinogenesis as the cells underwent the physiological changes associated with transformation was examined more precisely with 6-hr lymphotoxin treatments. Lymphotoxin treatment before irradiation and TPA caused a transient cellular change. When the cells were initiated within 2 days after lymphotoxin exposure, the induction of promoted transformation was inhibited. Results were similar with nonpromoted transformation. Lymphotoxin became a more effective anticarcinogen as the interval between the lymphotoxin pulse and carcinogen insult or TPA addition was reduced. When added during the last 6 hr of the experiment, lymphotoxin was equally inhibitory, whether or not TPA was present. Thus, lymphotoxin induces an anticarcinogenic physiological state that is short-lived or transient; the temporal relationship between lymphotoxin and carcinogen exposure is important for preventing initiated or promoted transformation.
Animals↗
Transformation of Bloom's syndrome fibroblasts by DNA transfection.
Nonmalignant diploid human fibroblast cells (GM3498B) derived from a skin biopsy of a patient with Bloom's syndrome have been transformed by transfection with DNA from a tumorigenic mouse cell line (Ha-8) carrying a single copy of the Harvey murine sarcoma virus (Ha-MuSV) genome. The transformed cell lines have an extended life-span, form colonies in agarose, and proliferate in nude mice--characteristics of neoplastic transformation. Like the parental cells, they also exhibit a high spontaneous level of sister chromatid exchanges. Finally, the transformed cells contain most, if not all, of the Ha-MuSV genome as well as the human rasH sequence. These experiments show that these diploid nonmalignant human cells can be used as recipients in transfection experiments for studying the genetic control of neoplastic transformation.
Bloom Syndrome↗
Initiation of carcinogenesis dependence upon MNNG-induced release of the G1 block of density-inhibited Syrian hamster cells.
After N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of density-inhibited postconfluent (DDIR) Syrian hamster embryo cell cultures, only a small population of the cells incorporate [3H]-thymidine within the next 10-20 h. The transformed colonies, observed subsequent to reseeding, are derived from the population incorporating [3H]-thymidine. This study demonstrates that MNNG-induced thymidine incorporation resulted from semiconservative DNA synthesis as analyzed by DNA density gradients. Furthermore, cell cycle distributions at selected times after treatment indicate that the MNNG-responsive population was released from the density-dependent G1 block, proceeded through S, and became blocked again at G2 or M. These results indicate that DNA synthesis is temporally related to an early step in carcinogenesis.
Animals↗
Nitration of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons results in products able to induce transformation of Syrian hamster cells.
Five nitrated polycyclic aromatic hydrocarbons, synthesized from benzo[a]pyrene, fluoranthene, pyrene, and chrysene induced dose-dependent transformation of Syrian hamster embryo cells. Benzo[a]pyrene, a known carcinogen, induced transformation while the other parental compounds, which are non-carcinogens, were not effective. The transforming potential of the nitro derivatives varied from compound to compound; on a molar basis, 1,8-dinitropyrene was the most effective nitrated hydrocarbon followed in order by 3-nitrofluoranthene, 1-nitropyrene, 6-nitrochrysene, and 6-nitrobenzo[a]pyrene. The ability to obtain dose-dependent transformation frequencies indicates that hamster cell transformation represents a responsive model for elucidating the mechanism of action of nitrated carcinogens. Because of their ubiquitous distribution and their ability to induce morphological transformation in mammalian cells, nitrated polycyclic aromatic hydrocarbons must be considered as potential carcinogens.
Animals↗
Asbestos and benzo(a)pyrene synergism in the transformation of Syrian hamster embryo cells.
Four varieties of asbestos fibers, crocidolite, anthophyllite, amosite, and chrysotile, induced a low rate of morphologic transformation in Syrian hamster cells. Of the four tested, chrysotile was the most lethal as reflected by colony survival. When cells were exposed to 1 microgram of benzo(a)pyrene (BP)/1 ml medium or 3 J/m2 ultraviolet irradiation and to different concentrations of the asbestos fibers, an enhancement of transformation occurred only with BP. The enhancement was dose responsive with all fiber species except for amosite which was dose independent. The synergistic activity of BP and asbestos suggests that asbestos facilitates the transport of BP to the cell site(s) critical for transformation. These results provide a basis for investigating the carcinogenic and cocarcinogenic potential of asbestos fibers in mammalian cells.
Animals↗
Phytohemagglutinin inhibits phorbol diester promotion of UV-irradiation initiated transformation in Syrian hamster embryo cells.
Phytohemagglutinin (PHA) or either of its isolectins, erythroagglutinin or leukoagglutinin, causes a dose-dependent decrease in 12-0-tetradecanoylphorbol 13-acetate (TPA)-promoted transformation of Syrian hamster embryo cells, but has no effect on transformation induced by ultraviolet irradiation. The ineffectiveness of concanavalin A indicates that not every lectin inhibits TPA. Galactose, a dominant sugar in receptors for PHA binding, reverses the inhibition of TPA promotion caused by PHA but galactose does not inhibit TPA promotion itself. Therefore, the TPA and PHA binding sites are functionally discrete. The PHA inhibition of TPA-promoted transformation is reversible because PHA is only effective if present with TPA, whereas lymphotoxin, an immunologic hormone, has a persistent anti-carcinogenic effect, regardless of whether it is added before or after TPA. PHA in conjunction with lymphotoxin causes additional inhibition of TPA-promoted transformation. PHA and lymphotoxin affect the biological activity of TPA by diverse mechanisms. Lymphotoxin alters the physiological state of the cell, causing a change in the cellular response to TPA. PHA may affect either the binding of TPA to a critical cellular receptor for promotion or a later step in promotion.
Agglutinins↗
Neoplastic transformation of Syrian hamster embryo cells by bisulfite is accompanied with a decrease in the number of functioning replicons.
Bisulfite, a chemical that at neutral pH does no induce mutations at two loci in Chinese hamster V-79 cells, induces transformation of Syrian hamster embryo cells. Although bisulfite affects DNA metabolism, there was no evidence of bisulfite-induced DNA damage. Bisulfite induced no excision repair replication, caused no DNA strand breaks detectable in alkaline sucrose gradients, had no effect on the size distribution of DNA nascent daughter strands, and did not affect excision on post-replication repair of u.v.-induced damage. However, bisulfite did induce a dose-dependent decrease in the rate of DNA replication per cell, apparently due to a reduction in the number of functioning replicons. Since the data indicate that bisulfite causes no detectable DNA damage, it appears that bisulfite induces transformation by a non-mutagenic event that could involve the inhibition of semi-conservative DNA synthesis.
Animals↗
Neoplastic transformation of Syrian hamster cells by putative epoxide metabolites of commercially utilized chloroalkenes.
The transforming ability of six epoxides of structurally related chloroalkenes was determined with a quantitative Syrian hamster embryo cell model. The epoxides used were cis-1-chloropropene oxide (c-CPO), trans-1-chloropropene oxide (t-CPO), cis-1,3-dichloropropene oxide, trans-1,3-dichloropropene oxide, trichloroethylene oxide (TCEO), and tetrachloroethylene oxide (PCEO). All six epoxides induced morphologic transformation of Syrian hamster embryo cells and caused cell lethality as reflected in the reduced cloning efficiency; in all instances, transformation was observed with less than 25% toxicity. The potency of the various epoxides was influenced by the difference in stability of the compounds. The results with c-CPO, t-CPO, TCEO, and PCEO were consistent with a linear dose response. The transformation results reflect the carcinogenicity of the parental chloroalkenes tested thus far. Furthermore, if the epoxides are metabolic intermediates of the chloropropene parent chloropropenes, the epoxides are probably proximate carcinogens.
Animals↗
Transformation of Syrian hamster embryo cells by sodium bisulfite.
Exposure at neutral pH of Syrian hamster embryo cells (HEC) to non-lethal, non-mutagenic doses of bisulfite, the physiological form of SO2, causes a dose-dependent increase in transformation. When bisulfite is used in combination with UV irradiation, the induction of transformation is not synergistic. These results suggest that bisulfite transformation of HEC may not occur by a mechanism involving mutation.
Animals↗