Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

scholarly journals Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098 ◽  
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

scholarly journals Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

1982 ◽  
Vol 2 (3) ◽  
pp. 308-319 ◽  
Author(s):  
G M Wahl ◽  
L Vitto ◽  
R A Padgett ◽  
G R Stark
Keyword(s):  
In Situ Hybridization ◽  
Syrian Hamster ◽  
Specific Radioactivity ◽  
Large Chromosome ◽  
Hybridization Technique ◽  
Wild Type ◽  
Aspartate Transcarbamylase ◽  
Metaphase Chromosomes ◽  
Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells

1982 ◽  
Vol 2 (3) ◽  
pp. 293-301
Author(s):  
R A Padgett ◽  
G M Wahl ◽  
G R Stark
Keyword(s):  
Electron Microscopy ◽  
Syrian Hamster ◽  
Specific Inhibitor ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Intervening Sequences ◽  
Multiple Copies ◽  
Cad Gene ◽  
Polyadenylated Rnas ◽  
Ump Synthesis

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

The Diterpenoid Lactone Andrographolide Induces Reactive Oxygen Species (ROS) Dependent Apoptosis in ATRA Sensitive and Resistant APL Cell Lines.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2446-2446
Author(s):  
Shuo Yang ◽  
Jessica K. Altman ◽  
Sheila Prachand ◽  
Austin Tom ◽  
Bo Ding ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Viability ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Annexin V ◽  
Resistant Cell ◽  
Oxygen Species ◽  
Resistant Lines ◽  
Sensitive Line

Abstract Abstract 2446 Andrographolide is a crystalline diterpenoid lactone. It consists of an α-alkylidene- g-butyrolactone moiety and three hydroxyls at C-3, C-14 and C-19, which are responsible for its biological activities. It is the major bioactive ingredient of the medicinal plant Andrographis paniculata and it has been used in Asia for a variety of non-malignant conditions. We previously reported that Andrographolide results in mitochondrial-mediated apoptosis in lymphoma cell lines and fresh malignant cells from patients with lymphoma (Yang et al. Clin Cancer Res 2010:16:4755). Based on the mechanism of action in lymphoma and a prior report in APL (Manikam et al. J Pharm Pharmacol 2009:61:9), we hypothesized that andrographolide may have biological activity in acute promyelocytic leukemia (APL) an that this may be related to reactive oxygen species (ROS). We therefore investigated the effects of andrographolide on cell viability, apoptosis induction, mitochondrial membrane poential and signaling pathways in 3 APL cell lines, the ATRA sensitive line NB4 and the ATRA-resistant lines NB4–007/6 and NB4–306 and 3 samples from patients with APL. Methods: NB4 (ATRA sensitive cell line), NB4–007/6 and NB4–306 (ATRA resistant cell lines) were cultured in RPMI-1640 under standard conditions. Cell viability was measured using the trypan blue or propidium iodide exclusion method. Fresh leukemic cells were obtained from 3 patients after informed consent according to an NU IRB approved protocol. One had ATRA-resistant APL and 2 had de-novo untreated APL. We measured apoptosis by Annexin V-FITC by FACS. We measured mitochondrial membrane potential and cell differentiation by standard techniques. Results: Incubation with increasing concentrations of andrographolide demonstrates loss of cell viability as measured by MTT assay. The IC50 at 48 hours was 6uM for NB4–306, 6.5uM for NB4–007/6 and 9uM for NB4. Apoptosis by Annexin V/FACS demonstrated that at 48 hours there was increasing apoptosis in all 3 cell lines and that the ATRA-resistant cell lines NB4–007/6 and NB4–306 were significantly more sensitive to andrographolide than the ATRA sensitive cell line NB4 (p< 0.025). This was accompanied by PARP and caspase 3-cleavage. There was evidence of decrease in mitochondrial membrane potential, but no effect on differentiation as measured by CD11b expression by flow. We next interrogated signaling pathways and found that in the ATRA resistant line NB4–007/6 there was an increase in phosphorylation of the Forkhead box O transcription factors p-FOXO1 at Thr24 and up-regulation of FasL (which peaked at 6 hours) and p27Kip1. We also demonstrated that andrographolide caused N-acetyl L- cysteine (NAC) reversible down regulation of c-MYC (in the ATRA resistant lines) and p-AKT (T308) (in the ATRA sensitive line) expression. In fresh patient specimens (n=3) there was dose dependent increase in apoptosis at 48 hours (>70% at 10uM, 85% at 20uM). From prior reports and our own data we suspected that the effects of andrographolide were dependent on reactive oxygen species (ROS), and indeed apoptosis was completely inhibited by NAC. Conclusion: Taken together, these data suggest that andrographolide, a novel natural diterpenoid lactone with significant biological activity in cancer, may have activity in patients with ATRA-resistant APL by a mechanism of action that is distinct from ATRA. We believe that these data provide a compelling rationale to add this natural diterpenoid lactone to the clinical trial agenda in APL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 293-301 ◽  
Author(s):  
R A Padgett ◽  
G M Wahl ◽  
G R Stark
Keyword(s):  
Electron Microscopy ◽  
Syrian Hamster ◽  
Specific Inhibitor ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
Intervening Sequences ◽  
Multiple Copies ◽  
Cad Gene ◽  
Polyadenylated Rnas ◽  
Ump Synthesis

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

scholarly journals ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942

Oncogene ◽  
2019 ◽  
Vol 39 (3) ◽  
pp. 651-663 ◽  
Author(s):  
Georgina P. Sava ◽  
Hailing Fan ◽  
Rosemary A. Fisher ◽  
Sabrina Lusvarghi ◽  
Sunil Pancholi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Cancer Therapeutics ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Common Mechanism ◽  
Mechanism Of Resistance ◽  
Resistant Lines ◽  
Resistant Cells

Abstract The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.

Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells

1983 ◽  
Vol 3 (3) ◽  
pp. 391-398
Author(s):  
I L Andrulis ◽  
C Duff ◽  
S Evans-Blackler ◽  
R Worton ◽  
L Siminovitch
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Asparagine Synthetase ◽  
Chromosome 1 ◽  
Ovary Cell ◽  
Synthetase Activity

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity

1983 ◽  
Vol 61 (2-3) ◽  
pp. 120-129 ◽  
Author(s):  
Robert G. Hards ◽  
Jim A. Wright
Keyword(s):  
Enzyme Activity ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Resistant Cell ◽  
Activity Levels ◽  
Drug Resistant ◽  
Enzyme Substrate ◽  
Resistant Lines

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.

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