scholarly journals Role and mechanism of miR-130a-3p in the chemosensitivity of retinoblastoma cells to vincristine

2021 ◽  
Author(s):  
Xiulan Lu ◽  
Huifang Tu ◽  
Dongrun Tang ◽  
Xiaoming Huang ◽  
Fengyuan Sun
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Resistance Index ◽  
Parental Cell ◽  
Luciferase Assay ◽  
Parental Cell Line ◽  
Pax6 Expression ◽  
Retinoblastoma Cells ◽  
Primary Obstacle

Abstract Purpose Chemoresistance remains the primary obstacle threatening the prognosis of retinoblastoma (RB). microRNAs (miRNAs) are acknowledged as critical regulator of drug resistance. This study explored the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to vincristine (VCR). Methods miR-130a-3p expression of VCR-sensitive and VCR-resistant RB tissues was detected using RT-qPCR. VCR-resistant RB cell line Y79/VCR was induced. miR-130a-3p expression of Y79/VCR cell line and its corresponding parental cell line was detected. Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The cell proliferation was measured using MTT assay, and the IC50 value and drug resistance index were examined using CCK-8 assay. The targeting relationship between miR-130a-3p and PAX6 was predicted through bioinformatics analysis and verified using dual-luciferase assay. Functional rescue experiments were conducted to confirm the role of PAX6 in chemosensitivity of RB cells. The effect of miR-130a-3p on tumorigenesis and VCR sensitivity was observed in vivo. Results miR-130a-3p was downregulated in VCR-resistant RB tissues and cells. Overexpression of miR-130a-3p repressed the proliferation of VCR-resistant RB cells and enhanced chemosensitivity. miR-130a-3p targeted PAX6 expression. Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of RB. Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo. Conclusion miR-130a-3p enhanced the chemosensitivity of RB cells to VCR by targeting PAX6 expression.

Loss of EZH2 and Aberrant DNA Methylation Account for PKC412 Drug Resistance in the FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cell Line MV4-11,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3468-3468
Author(s):  
Stefanie Göllner ◽  
Tino Schenk ◽  
Christian Rohde ◽  
Arthur Zelent ◽  
Friedrich Stölzel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Drug Resistance ◽  
Cell Line ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Protein Levels ◽  
Apoptosis Pathways

Abstract Abstract 3468 The development of drug resistance is a common feature in AML that occurs towards classical cytotoxic drugs as well as novel kinase inhibitors. Mutations in primary drug targets explain a significant fraction of acquired drug resistance but the mechanisms of resistance still remain unknown in most patients. About 20–30% of all AML patients possess the mutant FLT3-ITD leading to a constitutive activation of the FLT3 kinase. Flt3-mutations can be targeted by treatment with tyrosine kinase inhibitors. Resistance to these kinase inhibitors is an increasing phenomenon whose mechanisms are not entirely understood today. As epigenetic mechanisms are shown to play an important role in leukemia pathogenesis they are also likely to influence drug resistance. To elucidate the potential role of epigenetic mechanisms in the development of drug resistance towards kinase inhibitors we used a PKC412 partially resistant clone (MV4-11R) of the AML cell line MV4-11, which harbors a homozygous FLT3 internal tandem duplication (ITD) mutation. An initial screening for histone modifying enzymes revealed a downregulation of EZH2 on mRNA as well as protein level compared with the parental cell line. The reduction of EZH2, a H3K27 methyltransferase, in MV4-11R is furthermore correlating with globally diminished H3K27me3 levels. ChIP-Seq experiments using H3K27me3 antibody revealed differences in histone 3 K27 methylation at specific promoter sites between the parental and resistant MV4-11. To test for an increased drug resistance due to reduced EZH2 protein levels lentiviral knock-down of EZH2 was performed in the MV4-11 parental cell line and three individual knock-down cell clones were investigated for their drug resistance potential. These knock downs all showed elevated IC50 values as well as resistance towards the apoptosis-inducing effects of PKC412 compared with the scrambled shRNA cells. Furthermore, EZH2 protein levels of 5 FLT3-ITD-positive AML patient samples were determined by Western Blot, samples were treated with PKC412 for 3 days and cell survival was assayed. Using this approach higher EZH2 levels in patients could also be associated with a higher sensitivity to PKC412 pointing to a putative role of EZH2 in the development of PKC412 resistance in vivo. As EZH2 has been shown to interact with DNMTs in the context of the Polycomb Repressive Complex 2 and 3 (Viré et al., Nature 2006), we analyzed whether parental and resistant MV4-11 differ in their DNA methylation pattern. To identify hyper-/hypomethylated genes in MV4-11R, we applied the Illumina 27k Methylation BeadChip approach as well as Reduced Representation Bisulfite Sequencing (RRBS) for a genome wide CpG methylation analysis. In particular genes associated with apoptosis pathways and signal transduction were hypermethylated in MV4-11R cells compared to the parental cell line. Based on the observation of DNA methylation changes between the parental cell line and MV4-11R, treatment with the demethylating agent 2-deoxy-5-azacytidine (Aza dC) was conducted to investigate recovery of drug sensitivity. Incubation with 250 nM Aza dC for 5 days could restore the sensitivity of MV4-11R towards PKC412 as shown in proliferation and apoptosis assays. Using the Affymetrix Human Gene 1.0 ST Array platform we identified 110 genes whose expression was reactivated after treatment of MV4-11R with Aza dC, predominantly genes playing a role in signal transduction, apoptosis pathways and cell cycle. A cell cycle analysis of the MV4-11 and MV4-11R cells indeed showed that the resistant cell line is cycling less than the parental one, which possibly also favors the resistance development towards PKC412. Summarized, our data show that a loss of EZH2 accompanied by DNA methylation changes lead to PKC412 resistance in a FLT3-ITD AML cell line model possibly reflecting a way of acquisition of drug resistance in patients. Disclosures: Thiede: Novartis: Lectures, Research Funding. Müller-Tidow:Novartis: Research Funding.

miR-503-5p induces doxorubicin resistance in triple-negative breast cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1083-1083
Author(s):  
Iris Garrido-Cano ◽  
Anna Adam-Artigues ◽  
Ana Lameirinhas ◽  
Birlipta Pattanayak ◽  
Eduardo Tormo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Doxorubicin Resistance ◽  
Tnbc Cell Line ◽  
Gain Loss ◽  
Resistance To Doxorubicin

1083 Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype comprising approximately 15% of BC. Conventional cytotoxic chemotherapies continue to be the mainstay for treatment of this BC, which lacks targetable markers. In this context, microRNAs have been described to have an important role. The aim of this work was to elucidate the function of miR-503-5p in doxorubicin resistance in TNBC. Methods: miR-503-5p expression was evaluated in the TNBC cell line with acquired resistance to doxorubicin (MDA-MB-231R) and its parental cell line (MDA-MB-231), by qRT-PCR. Studies of gain/loss of function of miR-503-5p were carried out in MDA-MB-231 and MDA-MB-231R cells by transient transfection of mimics and inhibitors. Cells were treated with doxorubicin, and viability was measured by flow cytometry and MTT assay. The role of miR-503-5p was also evaluated in vivo by Chicken Chorioallantoic Membrane (CAM) assay. MDA-MB-231 cells transfected with miR-503-5p mimic or scramble miRNA were inoculated onto the CAM of fertilized chicken eggs. After 48 hours, tumours were treated with doxorubicin or supplemented media for 48 hours and tumour growth was measured. miR-503-5p expression was quantified by qRT-PCR in a retrospective cohort of 74 TNBC patients treated with anthracycline + taxane regimens. Overall survival analysis for miR-503-5p in TNBC patients from METABRIC dataset was evaluated by the KM plotter online tool. Results: miR-503-5p was significantly upregulated in the resistant MDA-MB-231R TNBC cell line when compared to its parental cell line MDA-MB-231 (̃3.5-fold; p< 0.0001). Then, gain/loss function assays showed that upregulation of miR-503-5p in MDA-MB-231 cells increased resistance to doxorubicin ( p< 0.0001) and its downregulation in MDA-MB-231R cells had the opposite effect ( p< 0.0001). Moreover, the role of miR-503-5p was also confirmed in the CAM assay in vivo model, where miR-503-5p overexpression inhibited the effect of doxorubicin. In our cohort of patients, miR-503-5p expression levels in core biopsies sampled before preoperative chemotherapy were associated with residual cancer burden (RCB). miR-503-5p expression was significantly higher in patients with poor response to chemotherapy (RCB II and III; median, 95% CI: 0.00055, 0.00024 - 0.00136) than in patients with good response (RCB 0 and I; median, 95% CI: 0.00018, 0.00011 - 0.00034; p = 0.036). Moreover, we confirmed that TNBC patients with high expression of miR-503-5p had worse overall survival than patients with low expression ( p= 0.016). Conclusions: We identified miR-503-5p as a modulator of doxorubicin resistance in TNBC. Our in vitro findings are supported by the clinical data of TNBC patients and in vivo assays. Hence, the inhibition of miR-503-5p may be a promising strategy to improve chemotherapeutic efficacy. Moreover, the expression levels of miR-503-5p may be used as a biomarker for therapy response in TNBC.

Stable expression of a human HSP70 gene in a rat myogenic cell line confers protection against endotoxin

1996 ◽  
Vol 270 (4) ◽  
pp. C1017-C1021 ◽  
Author(s):  
S. H. Chi ◽  
R. Mestril
Keyword(s):  
Heat Shock ◽  
Protective Effect ◽  
Cell Line ◽  
Cell Injury ◽  
Marker Gene ◽  
Parental Cell ◽  
Myogenic Cell ◽  
Parental Cell Line ◽  
Subsequent Exposure

Recent reports show that a pre-heat shock has a protective effect against endotoxin “in vivo” in rodents. It has remains unclear what actually confers the protection against endotoxin. One candidate for this protective effect is the heat shock protein of 70 kDa (HSP70). We found that a mild heat shock pretreatment is the rat myogenic cell line, H9c2(2-1), confers resistance to a subsequent exposure to endotoxin. A myogenic rat cell line stably transfected with the human inducible HSP70 exhibits an increased survival rate compared with cells stably transfected solely with the selectable neomycin marker gene or the parental cell line H9c2(2-1) when exposed to endotoxin. The mechanism of endotoxin-induced cell injury is postulated to be through the generation of nitric oxide in these myogenic cells during exposure to endotoxin. We conclude that HSP70, regardless of the particular mechanism of cytotoxicity, plays a role in protecting the cell against the deleterious effects of endotoxin.

scholarly journals Is Rhodamine 123 an Appropriate Fluorescent Probe to Assess P-Glycoprotein Mediated Multidrug Resistance in Vinblastine-Resistant CHO Cells?

1997 ◽  
Vol 14 (3) ◽  
pp. 129-140 ◽  
Author(s):  
Jordi Pétriz ◽  
José Enrique O’Connor ◽  
Mercè Carmona ◽  
Joan García‐López
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Parental Cell ◽  
High Reactivity ◽  
Rhodamine 123 ◽  
Parental Cell Line ◽  
Experimental Conditions ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Drug Resistant Phenotype

Cellular drug resistance, which involves several mechanisms such as P‐glycoprotein (P‐gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamine 123 (Rho123) efflux reveals accurately the drug‐resistant phenotype. To study cellular drug resistance, we have obtained a CHO‐K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P‐gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug‐sensitive parental cell line CHO‐K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO‐K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO‐K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P‐gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123‐based functional assays of drug resistance should be evaluated for each cellular experimental model.

scholarly journals Mutations in the ND2 Subunit of Mitochondrial Complex I Are Sufficient to Confer Increased Tumorigenic and Metastatic Potential to Cancer Cells

Cancers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1027 ◽  
Author(s):  
Joaquín Marco-Brualla ◽  
Sameer Al-Wasaby ◽  
Ruth Soler ◽  
Eduardo Romanos ◽  
Blanca Conde ◽  
...  
Keyword(s):  
Cell Line ◽  
Complex I ◽  
Metastatic Potential ◽  
Parental Cell ◽  
Metabolic Inhibitors ◽  
Parental Cell Line ◽  
Mhc I ◽  
Mtdna Mutations

Multiprotein complexes of the mitochondrial electron transport chain form associations to generate supercomplexes. The relationship between tumor cell ability to assemble mitochondrial supercomplexes, tumorigenesis and metastasis has not been studied thoroughly. The mitochondrial and metabolic differences between L929dt cells, which lost matrix attachment and MHC-I expression, and their parental cell line L929, were analyzed. L929dt cells have lower capacity to generate energy through OXPHOS and lower respiratory capacity than parental L929 cells. Most importantly, L929dt cells show defects in mitochondrial supercomplex assembly, especially in those that contain complex I. These defects correlate with mtDNA mutations in L929dt cells at the ND2 subunit of complex I and are accompanied by a glycolytic shift. In addition, L929dt cells show higher in vivo tumorigenic and metastatic potential than the parental cell line. Cybrids with L929dt mitochondria in L929 nuclear background reproduce all L929dt properties, demonstrating that mitochondrial mutations are responsible for the aggressive tumor phenotype. In spite of their higher tumorigenic potential, L929dt or mitochondrial L929dt cybrid cells are sensitive both in vitro and in vivo to the PDK1 inhibitor dichloroacetate, which favors OXPHOS, suggesting benefits for the use of metabolic inhibitors in the treatment of especially aggressive tumors.

scholarly journals Establishment of 5-Fluorouracil-resistant canine mammary tumor cell line

2017 ◽  
Vol 20 (1) ◽  
pp. 103-110 ◽  
Author(s):  
B. Zhou ◽  
D. Zhang ◽  
S. M. Pei ◽  
H. Zhang ◽  
H. C. Du ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Line ◽  
Mammary Tumor ◽  
Tumor Cell Line ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Canine Mammary Tumor ◽  
Mammary Tumor Cell ◽  
Mammary Tumor Cell Line

Abstract Canine mammary tumors are the most common neoplasms in intact female dogs. The surgery cannot always solve the problem, chemotherapy are recommend to these patients. However, chemotherapy could always fail because of multidrug resistance (MDR). Through stepwise increasing 5-Fluorouracil (5-FU) concentration in the culture medium, a 5-FU-resistant canine mammary tumor cell line CMT7364/5-FU was established to disclose the molecular mechanism of the drug resistance. Cell morphology, cell sensitivity to drugs, growth curves, expression of proteins, and chemo-sensitivity in vivo were compared between the parental cell line and resistant cell line. As compared it to its parental cell line (CMT7364), CMT7364/5-FU showed different morphology, cross-resistant to other chemo-drugs and a prolonged population doubling time (PDT). The drug efflux pump proteins (ABCB1 and ABCG2) in CMT7364/5-FU were up-regulated. In vivo, the similar result revealed that CMT7364/5-FU cell line was more resistant to 5-FU. In conclusion, a 5-FU-resistant canine mammary tumor cell line (CMT7364/5-FU) was successfully established, it can serve as a good model for researching the mechanism of MDR and screening effective agents to reverse drug resistance.

scholarly journals A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2051
Author(s):  
Caterina Peraldo-Neia ◽  
Annamaria Massa ◽  
Francesca Vita ◽  
Marco Basiricò ◽  
Chiara Raggi ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Intrahepatic Cholangiocarcinoma ◽  
Biological Behavior ◽  
Population Doubling ◽  
Sequencing Analysis ◽  
Tumor Type ◽  
Clinical Issue ◽  
Mesenchymal Transition

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

The Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine Treatment in Resistant 2D and 3D Model Triple Negative Breast Cancer Cell Line: ABCG2 Expression Data

2021 ◽  
Vol 21 ◽  
Author(s):  
Fatma Kubra Ata ◽  
Serap Yalcin
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profile ◽  
Three Dimensional ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Xtt Assay ◽  
Gene And Protein Expression

Background: Chemotherapeutics have been commonly used in cancer treatment. Objective: In this study, the effects of Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine have been evaluated on two-dimensional (2D) (sensitive and resistance) cell lines and three dimensional (3D) spheroid structure of MDA-MB-231. The 2D cell culture lacks a natural tissue-like structural so, using 3D cell culture has an important role in the development of effective drug testing models. Furthermore, we analyzed the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene and protein expression profile in this study. We aimed to establish a 3D breast cancer model that can mimic the in vivo 3D breast cancer microenvironment. Methods: The 3D spheroid structures were multiplied (globally) using the three-dimensional hanging drop method. The cultures of the parental cell line MDA-MB-231 served as the controls. After adding the drugs in different amounts we observed a clear and well-differentiated spheroid formation for 24 h. The viability and proliferation capacity of 2D (sensitive and resistant) cell lines and 3D spheroid cell treatment were assessed by the XTT assay. Results: Cisplatin, Irinotecan, 5-Fu, and Gemcitabine-resistant MDA-MB-231 cells were observed to begin to disintegrate in a three-dimensional clustered structure at 24 hours. Additionally, RT-PCR and protein assay showed overexpression of ABCG2 when compared to the parental cell line. Moreover, MDA-MB-231 cells grown in 3D showed decreased sensitivity to chemotherapeutics treatment. Conclusion: More resistance to chemotherapeutics and altered gene expression profile was shown in 3D cell cultures when compared with the 2D cells. These results might play an important role to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR in the 3D spheroid forms.

Regulation of c-met expression in B16 murine melanoma cells by melanocyte stimulating hormone

1999 ◽  
Vol 112 (5) ◽  
pp. 623-630
Author(s):  
D. Rusciano ◽  
P. Lorenzoni ◽  
M.M. Burger
Keyword(s):  
Melanoma Cells ◽  
Parental Cell ◽  
Melanocortin Receptor ◽  
Parental Cell Line ◽  
Melanocyte Stimulating Hormone ◽  
Murine Melanoma ◽  
The One ◽  
Murine Melanoma Cells ◽  
Stimulating Hormone

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(α) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line

1985 ◽  
Vol 5 (10) ◽  
pp. 2527-2532
Author(s):  
C H Jenh ◽  
P K Geyer ◽  
L F Johnson
Keyword(s):  
Cell Line ◽  
Thymidylate Synthase ◽  
Northern Blot Analysis ◽  
Mouse Cell ◽  
Parental Cell ◽  
S Phase ◽  
Parental Cell Line ◽  
Mouse Cell Line ◽  
Isolated Nuclei ◽  
Mrna Content

We studied the content and metabolism of thymidylate synthase mRNA in cultured mouse fibroblasts that were undergoing a serum-induced transition from the resting to growing state. The studies were performed with a 5-fluorodeoxyuridine-resistant 3T6 cell line (LU3-7) that over produces the enzyme and its mRNA about 50-fold and that regulates the expression of the thymidylate synthase gene in the same manner as the parental cell line. We have previously shown that the rate of synthesis of thymidylate synthase increases at least ninefold when the serum-stimulated cells traverse the S phase. Here we show, by Northern blot analysis, that thymidylate synthase mRNA increased 20- to 40-fold as cells progressed from resting to late S phase. About 85% of poly(A)+ thymidylate synthase mRNA was associated with polysomes at all times. The increase in thymidylate synthase poly(A)+ mRNA content was the result of an eightfold increase in the rate of production of this species, as shown by pulse-labeling studies. Pulse-chase analysis revealed that the half-life of thymidylate synthase poly(A)+ mRNA was similar in resting (9 h) and growing (7 h) cells. The rate of transcription of the thymidylate synthase gene, as determined in isolated nuclei, increased only by a factor of three to four during the S phase. Since the content of the message increased to a much greater extent than the rate of transcription of the gene, posttranscriptional controls must also play a role in regulating the content of thymidylate synthase mRNA under these conditions. Our results suggest that the cell may regulate the distribution of thymidylate synthase mRNA between a relatively stable poly(A)+ RNA species and a labile poly(A)- RNA species.

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