Chinese Hamster Lung Cell Line Research Articles

Induction of cell division disturbances in chinese hamster lung cell line V79 by a complex mixture of polycyclic aromatic hydrocarbons and aza-heterocyclic compounds

A complex chemical mixture of heavy oil (Z1003), composed of polycyclic aromatic hydrocarbons (PAHs) and aza-heterocyclic compounds (AHCs), has been analysed for its ability to induce cell division disturbances on Chinese hamster lung cell line V79. In addition to the complex chemical mixture (Z1003), separated fractions of PAHs and AHCs (including four subfractions according to their polarity) were investigated. The complex mixture Z1003, PAH and AHC fractions and subfractions (excluding the weak polar subfraction) caused a strong dose-dependent mitotic arrest in cell cultures of V79 cells. The increase of metaphases was accompanied by a decrease of anaphases and telophases. In low concentrations of Z1003, PAH and AHC fractions, predominantly initial metaphases (dislocated chromosomes and/or chromosomes arranged in groups) were observed. In higher concentrations, additionally full C-metaphases and metaphases with condensed chromatin were detected. The results indicate a partial or total loss of the spindle apparatus with the result of an aneuploidy-inducing potential. According to the linkage of aneuploidy to malignant cell transformation and cancer development, a carcinogenic potency of the complex chemical mixture Z1003 and its fractions by a non-genotoxic or aneugenic mechanism should be taken into consideration.

The possible role of acetyltransferase in the induction of cytogenetic effects by 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) in cultured Chinese hamster cells

When metabolically activated, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), a heterocyclic amine isolated from cooked food, is clastogenic in cultured Chinese hamster and human cells. Secondary metabolites of PhIP are formed via acetyltransferase (AT) and sulfotransferase (ST) activity; however, which is responsible for its clastogenic effect is unknown. We addressed this question. We used a parental Chinese hamster lung cell line and three sublines transfected with different AT genes to test the clastogenic (i.e., micronucleus-inducing) effects of metabolically activated PhIP and 7,12-dimethylbenz[ a]anthracene (DMBA) in the presence and absence of pentachlorophenol (PCP), a ST inhibitor. PhIP was significantly more clastogenic in the three AT-enriched sublines than in the parental line ( p < 0.001). DMBA (a ST-activated mutagen), on the other hand, equally induced MNs in all the cell lines. When PCP was added to the test system, the MN-induction ability of DMBA, but not of PhIP, decreased significantly ( p < 0.001). These findings strongly suggest that PhIP clastogenicity is due to AT activity and not to ST activity.

Analysis of micronucleus induction of pyrimethamine in in vitro CHL cells and in in vivo mouse bone marrow cells.

In vivo and in vitro mutagenicity of pyrimethamine were examined in the micronucleus test. Pyrimethamine strongly induced micronuclei in a dose-dependent manner in the in vitro micronucleus test using the Chinese hamster lung (CHL) cell line, when treated at 0.2-1.6 micrograms/ml for 48 h. The in vivo micronucleus test was carried out in mice after the first, second, third and fourth administration of doses up to 40 mg/kg p.o. The results showed no increased frequency of micronuclei after any treatment, though pyrimethamine was shown to persist at levels > 2 micrograms/ml in plasma after a single oral administration of 50 mg/kg.

The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells

Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3′ end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A) +mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3′ end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression.

Inhibitory Effect of Non-Dialyzable Extract of Spinach on the Induction of Chromosomal Aberrations in CHL Cells by Carcinogens.

The effect of the non-dialyzable extract of fresh spinach leaves on the induction of chromosomal aberrations by some carcinogens was examined in a cultured Chinese hamster lung cell line (CHL). The non-dialyzable fraction was found to suppress the induction of chromosomal aberrations such as chromatid exchanges and chromatid breaks by N-methyl-N'-nitro-N-nitrosoguanidine, hydrogen peroxide and mitomycin C.

A Chinese hamster fibroblast mutant defective in thrombin-induced signaling has a low level of phospholipase C-beta 1.

A mutant fibroblast, 2A4b, was isolated from the Chinese hamster lung cell line CCL39 by a previously described selection (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390) for cells deficient in thrombin-induced signaling. Although the antiporter activation by thrombin in 2A4b is only approximately 60% that in CCL39, the stimulation by serum is not significantly impaired, indicating that the defect in 2A4b lies upstream of the antiporter in the signaling pathway. The addition of thrombin to serum-starved 2A4b cells causes blunted responses both in production of inositol phosphates and in the cytosolic [Ca2+] transient, particularly when no Ca2+ is added to the external medium. The in vitro inositol phospholipid-specific phospholipase C (PLC) activity of 2A4b cytosol plus membrane extracts exceeds that in CCL39. However, immunoblots with antibodies to PLC isozymes show that although the levels of PLC-delta 1, PLC-gamma 1, and PLC-beta 3 are at least as great as those in CCL39, the amount of PLC-beta 1 in 2A4b is markedly deficient (< or = 10%). PLC-beta 1 is found primarily in the nucleus and in non-nuclear membranes of CCL39 and is proportionately low in these subcellular locations of 2A4b. Thrombin activation of phospholipases D and A2 is impaired in 2A4b. We postulate that the deficiency in PLC-beta 1 causes defective targeting of protein kinase C-alpha to specific membrane sites, which may be required for activation of these downstream phospholipases.

Selection for the expression of one form of Chinese hamster dihydrofolate reductase over another during growth in methotrexate

Two mammalian expression plasmids, each carrying a cDNA encoding a different allele of dihydrofolate reductase (DHFR) present in the Chinese hamster lung cell line DC-3F, were constructed. These simian virus 40 promoter-enhancerbased plasmids, designated pSVA75 and pSVMQ19, are identical except for a G→A transition at nucleotide 286 of the DHFR coding sequence. Due to this change, the enzyme expressed by pSVA75 contains Asp 95, while the enzyme expressed by pSVMQ19 has Asn 95 [Melera et al., J. Biol. Chem. (1988) 1978-1990]. Both forms of the enzyme are catalytically equivalent and are produced to similar levels in DC-3F cells [Yu and Melera, Cancer Res. (1993) 6031-6035; H.Y., A.H. and P.W.M., in preparation]. Clonal cell lines expressing one or the other DHFR allele were obtained via transfection of DHFR − Chinese hamster ovary cells, and 74 clones of each type isolated. These were pooled and divided into 40 aliquots, each of which was then subjected to selection by growth in sequentially increasing concentrations of methotrexate (MTX). Analysis of the resulting drug-resistant populations revealed that cells producing Asp 95 DHFR dominated with an overall frequency of 3:1, and therefore, under these growth conditions, display a selective advantage over those producing Asn 95 DHFR. These data extend previous observations showing that independent selections of the heterozygous parent cell line DC-3F in MTX result in threefold more MTX-resistant lines overexpressing the Asp 95-encoding DHFR allele than the Asn 95-encoding DHFR allele. Moreover, these results demonstrate that preferential selection for the expression of one DHFR allele over the other in DC-3F cells can be accounted for solely by differences in the enzymes themselves, a result consistent with recent biochemical studies demonstrating that the Asp 95 enzyme displays a dissociation constant for MTX that is 2.7-fold higher than the Asn 95 enzyme [Yu and Melera, Cancer Res. (1993) 6031–6035].

Coupled expression of Ca 2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system

Stable expression of a full-length cDNA encoding chicken fast muscle Ca 2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pHβFCaA3) in which the ATPase was cloned downstream of a human β-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca 2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12–16 and 4–6 μg per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca 2+ transport and ATPase partial reactions, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.

Construction of a dominant selectable marker using a novel dihydrofolate reductase

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu 22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978–1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu 22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38–48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539–1546]. Transfection of DHFR − Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR + phenotype with equal efficiency (i.e., > 2.5 × 10 −3 ), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR + cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of > 8 × 10 −4 . Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (88 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.

Clastogenicity of aporphine alkaloids in vitro.

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 19 aporphine alkaloids including apomorphine with and without rat liver homogenates (S9) mix. Eighteen of 19 alkaloids tested induced chromosomal aberration in the presence or absence of S9 mix. Among the alkaloids tested, anolobine, liriodenine and 4,5-dioxodehydrocrebanine induced chromosomal aberrations at relatively low concentrations and as low as 2.5, 5 and 3.13 micrograms/ml, respectively. Dicentrine, anolobine and 4,5-dioxodehydrocrebanine induced chromosomal aberrations with high frequencies. Apomorphine induced 10% aberrant cells at 12.5 micrograms/ml in the direct method and 12% at 100 micrograms/ml with S9 mix in the S9 method. Liriodenine which was the most potent mutagen for TA100 with S9 mix and roemerine which was the most potent for TA98 with S9 mix in Ames test were also clastogenic with and without S9 mix.

Full length and alternatively spliced pgp1 transcripts in multidrug-resistant Chinese hamster lung cells

In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts. Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product. A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e. human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function. Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165. Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein. S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines. Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 μg/ml of 1,3-DNP, 55% at 0.1 μg/ml of 1,6-DNP and 45% at 0.025 μg/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- > 1,6- > 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 μg/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.

Interaction of Amosite and Surface-Modified Amosite with a V79-4 (Chinese Hamster Lung) Cell Line

Interaction of Amosite and Surface-Modified Amosite with a V79-4 (Chinese Hamster Lung) Cell Line

Interaction of amosite and surface-modified amosite with a V79-4 (Chinese hamster lung) cell line.

We have been examining a number of chemically modified mineral fibers, derived from amosite asbestos, by in vitro methods to clarify the role of the fiber surface in determining biological activity. The various fibers have identical size distributions but differ in their affinities for components of the cell membrane. They were treated with boiling toluene or chemically modified by treatment with alkyldimethylchlorosilanes (R = C8, C18) that react with free-surface hydroxyl groups to form the corresponding siloxanes. Fibers in MEM supplemented with 15% fetal calf serum were added to a suspension of V79-4 cells labeled with tritiated thymidine and the mixture was incubated. Aliquots of this mixture were spun down on a density gradient to determine the degree of cell-fiber interaction. At 37 degrees C native amosite (UICC standard) stuck to cells within 15 min of incubation, and the amount of sticking was maximum within 70 min. Decreasing the temperature decreased the amount of sticking, and at 20 degrees C no sticking was observable. The chemically modified amosite and the amosite treated with boiling toluene did not stick to the cells even after 70 min. Soaking the toluene-treated amosite with aqueous solutions at room temperature for 48 hr produced a material that had the same sticking properties as the original untreated fiber. These results indicate that the silanol content, and possibly the degree of hydration of the fiber surface, is important for a fiber to stick to a cell surface.

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes: II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.

In vitro transformation of Chinese hamster lung cell line : Shibasaki Y, Ronne M. Institute of Anatomy and Cytology, Odense University, DK-5230 Odense M. Anticancer Res 1988;8:1285-9

In vitro transformation of Chinese hamster lung cell line : Shibasaki Y, Ronne M. Institute of Anatomy and Cytology, Odense University, DK-5230 Odense M. Anticancer Res 1988;8:1285-9

Reduced DNA topoisomerase II activity and drug-stimulated DNA cleavage in 9-hydroxyellipticine resistant cells

We have isolated a Chinese hamster lung cell line resistant to 9-hydroxyellipticine (DC-3F/9-OH-E) which is also cross-resistant to topoisomerase II inhibitors such as amsacrine and etoposide. In this work we have studied quantitatively both DNA topoisomerase II activity by decatenation of kinetoplast DNA and drug-stimulated DNA cleavage of pBR 322. DNA topoisomerase II activity of DC-3F/9-OH-E nuclear extract was reduced by 3.5-fold as compared to that from DC-3F (sensitive parent) nuclear extract. We also found that DC-3F/9-OH-E nuclear extracts have a reduced capacity to induce in vitro topoisomerase II-mediated DNA cleavage upon stimulation by etoposide and amsacrine (7- and 10-fold respectively). Besides, mixing nuclear extracts from both sensitive and resistant cells indicates that either the enzyme in resistant cells is modified or a modulating factor is associated to it. Our results suggest that the resistance of the DC-3F/9-OH-E cell line to topoisomerase II inhibitors might be due to both a reduced amount of the enzyme and its reduced ability to form the cleavable complex in the presence of drugs.

Antifolate-resistant Chinese hamster cells. Molecular basis for the biochemical and structural heterogeneity among dihydrofolate reductases produced by drug-sensitive and drug-resistant cell lines.

Nucleotide sequence analysis of a cDNA clone shown to direct the synthesis in Escherichia coli of a pI 6.5 form of dihydrofolate reductase (DHFR) with an apparent molecular weight of 21,000 has clarified the allelic nature of the DHFR genes present in the Chinese hamster lung cell line DC-3F. By comparison with other cDNAs encoding different forms of DHFR produced by these cells or by antifolate-resistant sublines derived from them (Melera, P.W., Davide, J.P., Hession, C.A., and Scotto, K.W (1984) Mol. Cell. Biol. 4, 38-48) and with the use of transcription vectors to generate homogeneous populations of specific DHFR mRNAs for subsequent translation in vitro, we demonstrate that, with respect to the proteins they encode, these alleles differ only at amino acid position 95; a conversion of Asp----Asn at this position is solely responsible for the electrophoretic mobility and pI differences between the Mr 21,000 pI 6.5 and Mr 20,000 pI 6.7 forms of the enzyme. We also show that the conversion of Leu to Phe at position 22 of the Mr 21,000 pI 6.5 enzyme results in a mutant form whose catalytic activity is equal to or greater than normal, but whose IC50 for methotrexate is 85 microM. Additionally, the in vitro translation experiments show that the minor pI forms of DHFR known to exist in Chinese hamster lung cells are generated by a translational or post-translational modification step. Preliminary evidence suggests that this modification may result from an acetylation of the N terminus of the protein.

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T. Van der Velde-Koerts, V. Ling, and P. Borst, Mol. Cell. Biol. 6:1671-1678, 1986). We report here that only a subset of these, encoding the 170-kilodalton P-glycoprotein, are consistently amplified in three different multidrug-resistant Chinese hamster lung cell lines, selected with vincristine, daunorubicin, or actinomycin D. Within each cell line, genomic sequences homologous to the P-glycoprotein cDNA probe were amplified to different levels. The pattern of differential amplification was consistent with the presence of at least two and possibly three P-glycoprotein genes. In the actinomycin D-selected cell line, these genes were disproportionately overexpressed relative to the associated levels of amplification. These results underline a central role for P-glycoprotein in multidrug resistance. In the daunorubicin-selected cell line, another, as yet uncharacterized, gene was amplified but disproportionately underexpressed. Its amplification was therefore fortuitous. We present a tentative map of the region in the hamster genome that is amplified in the multidrug-resistant cell lines which were analyzed.

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T. Van der Velde-Koerts, V. Ling, and P. Borst, Mol. Cell. Biol. 6:1671-1678, 1986). We report here that only a subset of these, encoding the 170-kilodalton P-glycoprotein, are consistently amplified in three different multidrug-resistant Chinese hamster lung cell lines, selected with vincristine, daunorubicin, or actinomycin D. Within each cell line, genomic sequences homologous to the P-glycoprotein cDNA probe were amplified to different levels. The pattern of differential amplification was consistent with the presence of at least two and possibly three P-glycoprotein genes. In the actinomycin D-selected cell line, these genes were disproportionately overexpressed relative to the associated levels of amplification. These results underline a central role for P-glycoprotein in multidrug resistance. In the daunorubicin-selected cell line, another, as yet uncharacterized, gene was amplified but disproportionately underexpressed. Its amplification was therefore fortuitous. We present a tentative map of the region in the hamster genome that is amplified in the multidrug-resistant cell lines which were analyzed.