Nicotine: potentially a multifunctional carcinogen?
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Carcinogens↗
Biomedical subjects
J A Campain
Publications and source records attributed to J A Campain.
Arsenic and benzo[a]pyrene differentially alter the capacity for differentiation and growth properties of primary human epidermal keratinocytes.
Normal human epidermal keratinocytes (NHEK) have been chosen as an in vitro model to test the hypothesis that chemicals which alter or interfere in cellular differentiation will concomitantly induce growth perturbations and are, thus, potential carcinogens. In these studies, we have focused on two known skin carcinogens, arsenic and benzo(a)pyrene (BaP). Our results demonstrated that BaP inhibits terminal differentiation in NHEK, as measured by cross-linked envelope (CLE) formation, up to 5.8-fold in control and 1.7-fold in calcium (Ca2+)-treated cells. In comparison, arsenic decreased CLE formation 20-fold in control cells and 5.5-fold in Ca2+-treated NHEK. To characterize the effects of these agents on the growth rate and cell cycle distributions of NHEK, flow cytometric analysis was used. BaP at 2 microM increased proliferation rates by 29%. Altered cell-cycle distribution in BaP-treated cells indicated a more rapid progression through the cell cycle, possibly by a shortened G2 phase. In contrast, arsenic at 5 microM inhibited proliferation by 25%; growth arrest (9%) was also observed in NHEK treated with 2 mM Ca2+. Our findings suggest that, although both BaP and arsenic inhibit CLE production in NHEK, different mechanisms may be involved. Studies in progress will attempt to identify molecular markers involved in the observed chemical effects. These markers will facilitate a mechanistic understanding of how an altered balance between growth and differentiation may play a role in the transformation process in NHEK.
Arsenic↗
Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes.
To evaluate health effects of chemical mixtures, such as multiple heavy metals in drinking water, we have been developing efficient and accurate hazard identification strategies. Thus, in this study, we determine the cytotoxicity of arsenic, cadmium, chromium, and lead, and characterize interactions among these metals in human epidermal keratinocytes. Three immortal keratinocyte cell lines (RHEK-1, HaCaT, and NM1) and primary keratinocytes (NHEK) were used. A statistical approach applying an additivity response surface methodology was used to test the validity of the additivity concept for a 4-metal mixture. Responses of the 4 keratinocyte strains to the metal mixture were highly dose-dependent. A growth stimulatory effect (hormesis) was observed in RHEK-1, NM1, and NHEK cells with the metal mixture at low concentrations (low ppb range). This hormesis effect was not significant in HaCaT. As the mixture concentration increased, a trend of additivity changed to synergistic cytotoxicity in all 4 cell strains. However, in NHEK, RHEK-1, and HaCaT, at the highest mixture concentrations tested, the responses to the metal mixtures were antagonistic. In NM1, no significant antagonistic interaction among the metals was observed. To explore a mechanistic basis for these differential sensitivities, levels of glutathione and metallothioneins I and II were determined in the keratinocyte cell strains. Initial data are consistent with the suggestion that synergistic cytotoxicity turned to antagonistic effects because at highest mixture exposure concentrations cellular defense mechanisms were enhanced.
Arsenic↗
Targeted gene delivery to Kaposi's sarcoma cells via the fibroblast growth factor receptor.
Kaposi's sarcoma (KS) is a major AIDS-related malignancy associated with significant morbidity and mortality. Current chemotherapeutic regimens are associated with a dismal prognosis. In an effort to develop a new approach to KS treatment, we devised a gene therapy-based adenovirus retargeting schema that redirects the adenovirus to fibroblast growth factor receptors endogenously present on the cell surface of KS cells. By using a bifunctional conjugate consisting of a blocking antiadenoviral knob Fab linked to basic fibroblast growth factor, FGF2, the gene transduction of KS cells was enhanced 7.7-44 fold; recombinant adenoviruses encoding either the firefly luciferase reporter gene, or the herpes simplex thymidine kinase gene, demonstrated quantitative enhancement of expression in the KS cell lines. In this regard, two KS cell lines that were previously refractory to native adenovirus transduction could be successfully transduced by the addition of the conjugate. This study thus addresses the utility of adenoviral retargeting to the FGF receptor in KS cells that are ordinarily transduction refractory to standardized approaches and allows practical development of gene therapy approaches for the treatment of human KS.
Acquired Immunodeficiency Syndrome↗
Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood.
Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.
Animals↗
Acquisition of multiple copies of a mutant topoisomerase IIalpha allele by chromosome 17 aneuploidy is associated with etoposide resistance in human melanoma cell lines.
Mutants of the human melanoma cell line, FEM-X, selected in multiple steps with VP- 16 (etoposide), are cross resistant to the epipodophyllotoxins and doxorubicin. Complementary DNA's for topoisomerase IIalpha were cloned from both FEM-X and FVP3, the most resistant mutant. Deletion of nucleotides 1320-1322 (or Ala429 from the resulting topoisomerase IIalpha protein) was unique to the cDNA from the drug resistant cell line. Expression of the mutant mRNA increases in parallel with VP-16 resistance in this series of cell lines. Restriction analysis and Southern analysis with allele-specific oligonucleotide probes were used to quantify the ratio of wild-type to mutant topoisomerase IIalpha alleles present in DNA amplified by PCR from both FEM-X and the drug resistant sublines. This analysis shows that in cell lines of increasing drug resistance, the number of mutant topoisomerase IIalpha alleles increases incrementally along with a concomitant decrease in the number of wild-type alleles. By quantitative Southern analysis of genomic DNA the total number of topoisomerase IIalpha alleles in FVP3 is approximately 2-fold that in the parental cells. Fluorescence in situ hybridization with a chromosome l7 paint reveals that amplification of the topoisomerase IIalpha locus in FVP3 correlates with an increase in the number of chromosome 17's, specifically the long arm. Cytogenetic analysis demonstrates that FEM-X contains three copies of chromosome 17, two of which are morphologically normal. During drug selection, FVP3 has gained 2-3 additional copies of the long arm of chromosome 17, the chromosomal location of the topoisomerase IIalpha locus. In this subline it is likely that three copies of the topoisomerase IIalpha gene are found on normal chromosome 17's and two on an isochromosome of the long arm of 17. By pulsed field gel electrophoresis, we were able to detect changes in the restriction pattern of the region of the long arm of chromosome 17 around the topoisomerase IIalpha locus that correlate with observed cytogenetic changes in FVP3. These results suggest that the acquisition of the mutant allele of topoisomerase IIalpha confers a selective advantage to cells in the presence of VP-16. As the drug concentration increased during the selection process, surviving sublines show preferential expression of the mutant topoisomerase IIalpha mRNA over that of the wild-type which is associated with a concomitant increase in the number of mutant topoisomerase IIalpha alleles.
Alleles↗
A novel mutant topoisomerase II alpha present in VP-16-resistant human melanoma cell lines has a deletion of alanine 429.
The human melanoma cell line FEM-X was selected in multiple steps with VP-16 (etoposide) and an inhibitor of P-glycoprotein (Campain et al., 1993). The resulting clones, FVP1b and FVP3, are highly resistant to the nonintercalative epipodophyllotoxins and exhibit moderate levels of resistance to doxorubicin. The topoisomerase II activity present in crude nuclear extracts from mutant and wild-type cells is similar in amount and equally sensitive to VP-16. However, in live cells, the topoisomerase II from FVP1b and FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. Using reverse transcription followed by the polymerase chain reaction (RT-PCR), we have cloned and sequenced the entire cDNA for topoisomerase II alpha from FEM-X and FVP3. The only sequence change unique to the cDNA from drug-resistant cells is a 3 bp deletion of nucleotide 1320-1322, resulting in a deletion of Ala429. Three FEM-X sublines of increasing resistance were tested, and the prevalence of the mutant RNA over wild-type increases in these cells in parallel with their resistance to VP-16. In FVP3, the most highly resistant line, expression of the wild-type allele is barely detectable. Analysis of genomic DNA shows that FEM-X is homozygous for the wild-type topoisomerase II alpha sequence and that each of the drug-resistant clones possesses both wild-type and mutant alleles. Although not definitive, these genetic results suggest that the deletion of Ala429 from topoisomerase II alpha makes the enzyme less susceptible to drug-induced cleavable complex formation and confers a growth advantage upon cells in the presence of VP-16.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Permissive effect of cyclic AMP and cycloheximide for induction by sodium butyrate of the glycoprotein hormone alpha-subunit gene in choriocarcinoma cells.
Expression of the chorionic gonadotropin alpha-subunit (alpha CG) gene is regulated differently in human tumor cell lines derived from trophoblastic and nontrophoblastic tissues. This is based, at least in part, on the observations that production of alpha CG is increased by cAMP but not by sodium butyrate in choriocarcinoma cells (JEG-3), whereas production of alpha CG is increased by butyrate but not by cAMP in cervical carcinoma cells (HeLa). Data presented herein confirm that the steady-state levels of alpha CG mRNA are increased approximately 10-fold by cAMP in JEG-3 cells, but additionally demonstrate that these levels can be further increased (to 25-fold) by sodium butyrate in conjunction with the cyclic nucleotide. Similar effects were achieved with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate, which also acted synergistically with cAMP to increase alpha CG mRNA levels (24-fold). Butyrate and 12-0-tetradecanoylphorbol-13-acetate had little or no effect when added alone (1.5-fold and 2.5-fold, respectively) or in combination with one another (3.8-fold). Induction of the alpha CG gene by cAMP was independent of protein synthesis, as mRNA levels were comparable when JEG-3 cells were treated with cAMP in the absence and presence of cycloheximide (CHX). Unexpectedly, alpha CG mRNA levels were also elevated 8- to 10-fold in response to a combination of butyrate and CHX, but CHX alone or in combination with 12-0-tetradecanoylphorbol-13-acetate had no effect. Gel mobility shift assays demonstrated changes in the pattern of proteins binding to a cAMP response element and to a trophoblast-specific element with nuclear extracts from cells treated with CHX but not with cAMP or sodium butyrate. Together, these results suggest that induction patterns of the alpha CG gene by butyrate and cAMP in trophoblast- and nontrophoblast-derived tumor cell lines are less distinct than suggested previously and indicate that CHX, which is permissive for induction by butyrate, causes a significant shift in electrophoretic mobility of trans-acting factors involved in alpha CG gene expression.
Base Sequence↗
Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II.
The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To characterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11-2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to four-fold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrates vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addition, the topoisomerase II present in nuclear extracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1) inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Differential DNA methylation of the chorionic gonadotropin beta-subunit multigene family.
In addition to its synthesis in the developing placenta, human CG or the isolated alpha- and beta-subunits are synthesized by a wide variety of both trophoblastic and nontrophoblastic tumor cell lines. The beta-subunit confers unique biological activity on the hormone and is encoded by a family of six genes or pseudogenes linked physically with beta LH at one locus on chromosome 19. Previous work has demonstrated that members of the beta CG family are not expressed to the same extent in first-trimester placenta. The factors allowing differential expression of the six nearly identical genes are unknown. It is also undetermined which of the multiple beta CG genes are actively expressed in the tumor cell lines that produce beta-subunit ectopically. One potential mechanism for control of beta CG gene expression is DNA methylation. A unique method of two-dimensional DNA electrophoresis was used to analyze the methylation status of each of the individual beta CG genes in placenta and in tumor cell lines that either do or do not express the protein. The DNA from each source exhibited unique, reproducible methylation patterns over the six beta CG genes. Particularly interesting were the observations that: 1) despite their nearly identical sequences and close chromosomal proximity, the six beta CG genes are distinguished from one another within the same and different cell types by their extent and pattern of methylation; 2) the beta CG locus is significantly hypomethylated in placenta, a state maintained in choriocarcinoma cells (JAr); 3) the beta CG gene cluster is unmethylated at a limited set of sites in DNA from both 2RA (a transformed fibroblast cell line that does not produce beta-subunit) and CBT (a glioblastoma cell line that produces beta-subunit ectopically), suggesting that general demethylation of the domain does not accompany activation of the locus in CBT but rather that site-specific changes may be responsible, at least in part, for inappropriate beta CG expression; and 4) the ratio of beta CG gene 3 transcripts to beta CG gene 5 transcripts is at least 10-fold higher in CBT cells than in JAr cells, suggesting that the reduced methylation in CBT cells of gene 3 compared to gene 5 may be a factor contributing to the activity of these genes.
Base Sequence↗
Deoxyribonuclease-hypersensitive sites in the glycoprotein hormone alpha-subunit gene from trophoblastic and nontrophoblastic human tumor cell lines: correlation with expression and effect of chemical inducers.
Human CG is composed of two subunits, alpha and beta. In addition to its eutopic synthesis in normal and malignant trophoblasts, the hormone is produced ectopically by a variety of tumor cell lines of nonplacental origin. Regulation of the alpha CG gene in trophoblasts appears to differ from that in nontrophoblasts. To determine whether these differences are reflected in the chromatin structure at the alpha CG locus, DNase I-hypersensitive sites within this domain were mapped in human tumor cell lines that differentially express the gene. Two hypersensitive sites were detected in DNA from cell lines that produce the alpha-subunit. The latter includes trophoblastic (JAr and JEG-3 choriocarcinoma) and nontrophoblastic (HeLa cervical carcinoma and ChaGo bronchogenic carcinoma) tumor cell lines. The most prominent site (HS 1) was located approximately 100 base pairs upstream from the transcription start site. In trophoblasts, accessibility of HS 1 increased substantially upon induction of the gene by cAMP, likely reflecting alterations in DNA-protein interactions at the cAMP response element and/or tissue-specific enhancer. In nontrophoblasts, where alpha-subunit synthesis is enhanced by sodium butyrate but not by cAMP, neither butyrate nor cAMP altered the accessibility of HS 1. The HS 2 is comprised of multiple sites with weak to moderate DNase sensitivity located downstream at +1600 to +4000 in cell lines that produce alpha-subunit. Cell lines that do not express the alpha CG gene possess a distinct hypersensitive site (HS 3) within the first intron at about +600; these include 3A-Sub-E (SV40 transformed placenta), CBT (glioblastoma multiforme), and CaSki (cervical carcinoma). Cleavage by DNase at HS 1 and HS 2 is not evident in nuclei from cell lines that do not produce alpha-subunit. These results suggest that HS 1 and HS 3 are characteristic of active and inactive states of the alpha CG gene, respectively, and that the accessibility of HS 1 generally correlates with the level of expression.
Cell Transformation, Neoplastic↗
Chorionic gonadotropin synthesis by human tumor cell lines: examination of subunit accumulation, steady-state levels of mRNA, and gene structure.
A number of tumor cell lines have been examined that differentially produce human chorionic gonadotropin and the isolated alpha- or beta-subunits. It has been demonstrated that all of the cell lines studied to date contain genes for both alpha- and beta-subunits, indicating that differential and exclusive expression of one subunit is not the result of a particular cell line having lost the gene for the alternate subunit as a consequence of chromosome changes accompanying cell transformation. Because many of these established cell lines are aneuploid, it is also significant that no evidence was found for gene amplification in cell lines producing alpha-subunit at very high levels compared to those with very low level expression. Analysis of restriction endonuclease digests of tumor cell DNAs has demonstrated identical patterns for beta-subunit in KpnI digests and KpnI/HindIII double digests. Polymorphisms were observed for alpha-subunit in EcoRI and HindIII digests, but these did not correspond with expression of the alpha-subunit. Significant levels of either mRNA (as determined by dot blot and Northern transfer hybridization analysis) were accompanied by corresponding elevated levels of alpha- and beta-subunits (as determined by radioimmunoassay), suggesting that regulation of subunit production most likely occurs at a pretranslational stage. However, there were apparent differences in the relative ratio of alpha- and beta-subunits and their cognate mRNAs among the cell lines.
Chorionic Gonadotropin↗
Lipid- and adenoviral-mediated gene transfer into AIDS-Kaposi's sarcoma cell lines.
Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.
AIDS-Related Opportunistic Infections↗