Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.     

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene.

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells.     

Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells.

The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

Linkage of early-onset osteoarthritis and chondrocalcinosis to human chromosome 8q.

Calcium pyrophosphate-deposition disease (CPDD), also called "chondrocalcinosis" or "pseudogout," is a disorder characterized by the deposition of calcium-containing crystals in joint tissue, which leads to arthritis-like symptoms. The presence of these crystals in joint tissue is a common finding in the elderly, and, in this population, there is a poor correlation with joint pain. In contrast, early-onset CPDD has been described in several large families in which the disease progresses to severe degenerative osteoarthritis (OA). In these families, an autosomal dominant mode of inheritance is observed, with an age at onset between the 2d and 5th decades of life. In this report, we describe a large New England family with early-onset CPDD and severe degenerative OA. We found genetic linkage between the disease in this family and chromosome 8q, with a multipoint lod score of 4.06. These results suggest that a defective gene at this location causes the disease in this family.     

Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression.

Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.