scholarly journals Certain changes in ornithine decarboxylase gene methylation accompany gene amplification

1991 ◽  
Vol 279 (2) ◽  
pp. 435-440 ◽  
Author(s):  
J Wahlfors
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Myeloma Cell ◽  
Parental Cell ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Parental Cell Line ◽  
Flanking Region

The ornithine decarboxylase (ODC; EC 4.1.1.17) gene in parental, dexamethasone-resistant and 2-difluoromethylornithine (DFMO)-resistant human IgG-myeloma-cell lines was studied with the aid of methylation-sensitive restriction endonucleases and probes recognizing different parts of the gene. In all cell lines the promoter region of the ODC gene appeared to be heavily methylated, whereas the first long intron was unmethylated. Methylation analyses of several clones from the parental cell line revealed that these cells are heterogeneous with respect to the methylation status of the ODC gene, whereas all clones from DFMO-resistant cell lines displayed the same methylation pattern. Two of the parental clones represented a hypomethylated type very close to that exclusively found among the DFMO-resistant clones with ODC gene amplification. This typical methylation pattern was due to decreased methylation of a few CCGG sequences in the 3′-flanking region of the gene. It is possible that this kind of hypomethylation favours the initiation of the gene-amplification process in certain individual cells. This hypothesis was supported by the finding that no hypomethylation was present in the ODC gene of another human myeloma cell line that had acquired resistance to DFMO without gene amplification. In a dexamethasone-resistant cell line that overproduced ODC mRNA at normal gene dosage there were some minor differences between the methylation pattern of the ODC gene of different clones, but no such hypomethylation could be found in clones from the parental cell line. In dexamethasone-resistant cells the ODC gene was hypomethylated around the two HpaII sites and three CfoI sites in the coding region and also, as well as in cells with amplified ODC sequences, in the 3′-flanking region of the gene. Some hypomethylation in the distant 5′-flanking region was also observed.

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.

A Novel Microarray Gene Expression Analysis of Murine AML Cell Lines Provides Clues to the Development of Drug Resistance to Ara-C

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5017-5017
Author(s):  
Susan K Rathe ◽  
David Largaespada
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Ribosomal Subunit ◽  
Nuclear Factor Κb ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Drug Resistant ◽  
Microarray Gene Expression ◽  
Expression Microarrays

Abstract Acute myeloid leukemia (AML) has the ability to evade cell death in the presence of chemotherapeutic cocktails containing cytosine arabinoside (Ara-C). This lab previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. B117H and B140H can tolerate Ara-C concentrations ~1000X that of their drug sensitive parental cell lines. mRNA from all four cell lines were used in gene expression microarrays for the purpose of comparing Ara-C drug resistant murine AML cell lines with their Ara-C drug sensitive parental lines. A novel algorithm was developed to evaluate the changes in gene expression between the drug resistant and drug sensitive cells. The algorithm differed from more conventional algorithms in two key ways. First, the detection data was normalized by using ribosomal subunit 9 (Rsp9) as the normalization gene, and secondly it calculated fold change by comparing the minimum value of one population to the maximum value of the other population. The output of this algorithm was a list of genes with significant gene expression changes. These genes were next submitted to the Ingenuity Pathway Analysis (IPA) process. IPA implicated nuclear factor-κB (NFκB) in the Ara-C resistance process. Cell growth assays confirmed that the Ara-C drug resistant B117H cell line was significantly more sensitive to NFκB inhibition than its Ara-C sensitive parental cell line. This leads us to believe that the selection of Ara-C resistance may also concomitantly make some AML cells highly sensitive to killing by NFκB inhibition. This theory is being tested further through the use of drug combination assays, to determine if a synergistic or antagonistic relationship exists between Ara-C and various drugs that affect the NFκB pathway.

scholarly journals Cytogenetic Evolution of Human Ovarian Cell Lines Associated with Chemoresistance and Loss of Tumorigenicity

2003 ◽  
Vol 25 (3) ◽  
pp. 115-122 ◽  
Author(s):  
Stéphanie Struski ◽  
Martine Doco‐Fenzy ◽  
Michael Koehler ◽  
Ilse Chudoba ◽  
Francis Levy ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chromosomal Abnormalities ◽  
Parental Cell ◽  
Comparative Genomic ◽  
Parental Cell Line ◽  
Multicolor Fish ◽  
Ovarian Cell ◽  
Genomic Changes ◽  
Ovarian Cell Lines

In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex‐FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug‐resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)). Colour figure can be viewed onhttp://www.esacp.org/acp/2003/25‐3/struski.htm.

Mouse Myeloma Cell Line Sp2/0 Multidrug-Resistant Variant as Parental Cell Line for Hybridoma Construction

2003 ◽  
Vol 22 (5) ◽  
pp. 321-327 ◽  
Author(s):  
Marina B. Melixetian ◽  
Maria A. Pavlenko ◽  
Elena V. Beriozkina ◽  
Zoya V. Kovaleva ◽  
Elena A. Sorokina ◽  
...  
Keyword(s):  
Cell Line ◽  
Myeloma Cell ◽  
Multidrug Resistant ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Myeloma Cell Line ◽  
Resistant Variant ◽  
Mouse Myeloma Cell ◽  
Mouse Myeloma Cell Line ◽  
Mouse Myeloma

scholarly journals Targeting of Deregulated Wnt/β-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines

2021 ◽  
Vol 22 (8) ◽  
pp. 4263
Author(s):  
Silvia Schmidtova ◽  
Katarina Kalavska ◽  
Veronika Liskova ◽  
Jana Plava ◽  
Svetlana Miklikova ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Treatment Options ◽  
Germ Cell Tumors ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Testicular Germ Cell ◽  
Apoptotic Effect ◽  
Pathway Inhibition

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/β-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of β-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of β-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/β-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/β-catenin pathway inhibition in GCTs is therefore warranted.

scholarly journals Understanding the Role of Extracellular Vesicles in Lenalidomide-Resistance Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1887-1887
Author(s):  
Tomofumi Yamamoto ◽  
Nobuyoshi Kosaka ◽  
Ochiya Takahiro ◽  
Yutaka Hattori
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Parental Cell ◽  
Resistant Cell ◽  
Bone Marrow Microenvironment ◽  
Drug Resistant ◽  
Apoptosis Assay ◽  
Drug Resistant Cell

Abstract Multiple myeloma (MM) is a plasma cell malignancy that develops by the accumulation of various genetic abnormalities. In recent years, the prognosis of MM has improved by the development of novel drugs including immunomodulatory drugs (IMiDs), proteasome inhibitors, and antibody drugs. However, MM cells acquired drug resistance by long-term exposure to these therapeutic drugs. MM is a multiclonal disease, and various clone subtypes develop within the bone marrow microenvironment. It has been suggested that drug resistant phenotype could transmit from resistant clones to sensitive ones, and also to immune cells, or mesenchymal stem cells, resulting in the change of bone marrow microenvironment suitable for MM cell survival. It has been shown that extracellular vesicles (EVs) are one of the means of information transmission. EVs are secreted from almost all cells, and the amount of EV secretion is particular high from cancer cells. It was already known that cancer-derived EVs transmitted information associated with cancer progressions such as angiogenesis, metastasis, and drug resistance to the surrounding cells. Thus, EVs were proposed to play an important role in acquisition of drug resistance even though the mechanisms have not been fully understood in MM. In order to understand the mechanism of drug resistance in MM mediated by EVs, lenalidomide resistant cell lines were established by long-term exposure of lenalidomide. The amount of EVs was measured by ExoScreen, which is ultra-sensitive detection method of EVs by measuring surface protein of EVs, such as, CD9 and CD63, and by the nanoparticle tracking analysis. We found that lenalidomide resistant cell lines in KMS21R, KMS27R and KMS34R cell lines secreted about twice more EVs than their parental cell lines, and the amount of EV secretion was correlated with the drug sensitivity of lenalidomide. Suppression of EV secretion by knockdown of TSG101, which is known for EV secretion-associated protein, did not affect lenalidomide resistance. We could suppress the EV secretion to two-thirds, however cell proliferation and caspase activity were not change. From these results, we postulated the two possibilities; 1) EV secretion pathway other than TSG101 is associated with drug resistance via EVs; 2) EV derived from lenalidomide resistant cells can affect the cells exist in bone marrow microenvironment. From these hypotheses, we have done the following experiments. Firstly, to identify the genes which involved in EV secretion pathway associated with drug resistance, RNA sequence among the drug-resistant cell lines and their parental cell lines was performed. Drug resistant cell lines had some genetic abnormality, for instance immune system or angiogenesis. Now, we are detecting the EV secretion associated genes in drug resistant cell lines. Secondly, EV derived from the drug-resistant cell lines and EV derived from the parent cell lines were added to drug sensitive MM cell lines, then lenalidomide is added after 24hr. The cell proliferation and apoptosis assay were evaluated after 48hr. EV derived from the drug-resistant cell lines in KMS34R cell line significantly inhibited cell death measured by MTS assay and apoptosis assay compared with those from the drug sensitive KMS21 and KMS34 cell lines. EVs from KMS34R cell line, which is the most progressed cell line we established, could more transmit drug resistance than those from other cell lines. These results suggested that drug resistance was transmitted from drug-resistant cell lines to non-resistant cell lines via EVs. Now, we are analyzing the component of EV from drug-resistant MM cells by proteome analysis to identify the molecules associated with lenalidomide resistance in MM. In addition, we are investigating the molecules which associated with the secretion of EVs from drug-resistance MM. These results prompted us to hypothesize that attenuating the function of a molecule responsible for EV secretion could lead to the inhibition of cancer development such as drug resistance. It is expected that EVs will be novel therapeutic targets in refractory or relapsed MM. Disclosures Hattori: IDAC inc.: Research Funding; Takeda: Research Funding.

Analysis of Intratumor Heterogeneity in Multiple Myeloma Using Methylcellulose Clonogenic Assays.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2346-2346
Author(s):  
Bonnie K. Arendt ◽  
Raphael Fonseca ◽  
Gregory Ahmann ◽  
Diane F. Jelinek
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Growth Regulation ◽  
Myeloma Cell ◽  
Intratumor Heterogeneity ◽  
Parental Cell Line ◽  
Myeloma Cells ◽  
Igf I

Abstract Multiple myeloma (MM) is a fatal disease characterized by the accumulation of malignant plasma cells in the bone marrow. Although progress has been made in better understanding growth control of this disease, effective treatment of MM patients with this disease is likely complicated by the extensive patient to patient variability that exists, as well variability within the tumor population itself. Thus, there is abundant evidence that intraclonal or intratumor heterogeneity exists in myeloma as revealed by morphologic and phenotypic heterogeneity in primary myeloma cells isolated from a single patient. We have had a long-standing interest in the growth regulation of myeloma cells and have hypothesized, along with other investigators, that there may only be a subset of myeloma cells that exhibits extensive proliferative potential. Understanding how cellular compartments within the malignant clone, as defined by identical immunoglobulin variable region sequence, may vary in growth regulation properties in isolation or in the company of less proliferative tumor cell subsets is key to understanding disease progression and how to better target the putative proliferative subset in myeloma. In this study, we have used a methylcellulose clonogenic assay to study intraclonal heterogeneity in a panel of human MM cell lines. Each of these cell lines, DP-6, KAS-6/1, KP-6, and, exhibit a variable response to IL-6 and IGF-I, and our goal was to evaluate growth responsiveness of individual subclones from each of these cell lines. Myeloma cell lines were plated at a concentration of 200-1000 cells in 1 ml Methocult H4533 in 35 mm gridded dishes with or without various cytokines. Following 3 weeks of culture, colonies were scored and those consisting of >40 cells were isolated, expanded, and studied further. Of interest, subclones isolated from each of the cell lines displayed significant differences in growth response to various cytokines in addition to specific morphologic and phenotypic differences. In this regard, results emerging from the DP-6 cell line were particularly intriguing. We have previously shown that the DP-6 cell line displays a proliferative response to both IL-6 and IGF-I and expresses autocrine IL-6 at a low level. Analysis of the growth properties of individual DP-6 clones revealed the existence of DP-6 cells (clone 1-15) that proliferate at a rapid rate in the apparent absence of exogenous growth factors. Whereas a neutralizing antibody to IL-6 did not inhibit cell growth, addition of a blocking antibody to the IGF-IR, (αIR3), completely blocked growth factor independent proliferation. Phenotypic analysis also displayed variation between the parental cell line and its subclone. For instance, the parental DP-6 cells largely expressed CD45 at a high level, whereas the clone 1-15 did not. Finally, we have also further characterized MM cell line subclones by gene profiling and FISH (fluorescence in situ hybridization) analysis to link specific phenotype and genotypes with patterns of cell growth. These results provide additional evidence that intratumor heterogeneity exists in myeloma. These studies further demonstrate how growth regulation may vary considerably among cellular subsets of the malignant population. Understanding what factors regulate the balance of specific myeloma cell subpopulations is key to an understanding of tumor progression. In summary, these studies provide a necessary foundation for future studies of the growth potential of subsets found in primary MGUS, SMM and MM patient samples.

scholarly journals Pharmacological Inhibition of PP2A Overcomes Nab-Paclitaxel Resistance by Downregulating MCL1 in Esophageal Squamous Cell Carcinoma (ESCC)

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4766
Author(s):  
Qi Song ◽  
Herui Wang ◽  
Dongxian Jiang ◽  
Chen Xu ◽  
Jing Cui ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Tumor Growth ◽  
Cell Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Squamous Cell ◽  
Resistant Cell ◽  
Parental Cell Line ◽  
Pharmacological Inhibition

Paclitaxel-based chemotherapy is a treatment option for advanced esophageal squamous cell carcinoma (ESCC). However, the development of chemoresistance leads to treatment failure, and the underlying mechanism remains elusive. We investigated the mechanisms of nanoparticle albumin-bound paclitaxel (nab-PTX) resistance by establishing three nab-PTX resistant ESCC cell lines. Proteomics analysis revealed higher oxidative phosphorylation (OXPHOS) in resistant cell line DR150 than in its parental cell line KYSE150, which is likely caused by stabilized anti-apoptotic protein MCL1. Additionally, we discovered the elevated activity of protein phosphatase 2A (PP2A), the phosphatase that dephosphorylates and stabilizes MCL1, in nab-PTX resistant cell lines. Pharmacological inhibition of PP2A with small molecule compound LB-100 decreased MCL1 protein level, caused more apoptosis in nab-PTX resistant ESCC cell lines than in the parental cells in vitro, and significantly inhibited the tumor growth of nab-PTX resistant xenografts in vivo. Moreover, LB-100 pretreatment partially restored nab-PTX sensitivity in the resistant cell lines and synergistically inhibited the tumor growth of nab-PTX resistant xenografts with nab-PTX. In summary, our study identifies a novel mechanism whereby elevated PP2A activity stabilizes MCL1 protein, increases OXPHOS, and confers nab-PTX resistance, suggesting that targeting PP2A is a potential strategy for reversing nab-PTX resistance in patients with advanced ESCC.

scholarly journals Replication of Toxoplasma gondii, but NotTrypanosoma cruzi, Is Regulated in Human Fibroblasts Activated with Gamma Interferon: Requirement of a Functional JAK/STAT Pathway

1999 ◽  
Vol 67 (5) ◽  
pp. 2233-2240 ◽  
Author(s):  
Isabela Penna Cerávolo ◽  
Andréa C. L. Chaves ◽  
Cláudio A. Bonjardim ◽  
David Sibley ◽  
Alvaro J. Romanha ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Cell ◽  
Human Fibroblasts ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Tryptophan Degradation ◽  
Parasite Replication ◽  
Stat Pathway

ABSTRACT To study the role of tryptophan degradation by indoleamine 2,3-dioxygenase (INDO) in the control of Trypanosoma cruzior Toxoplasma gondii replication, we used human fibroblasts and a fibrosarcoma cell line (2C4). The cells were cultured in the presence or absence of recombinant gamma interferon (rIFN-γ) and/or recombinant tumor necrosis factor alpha (rTNF-α) for 24 h and were then infected with either T. cruzi or T. gondii. Intracellular parasite replication was evaluated 24 or 48 h after infection. Treatment with rIFN-γ and/or rTNF-α had no inhibitory effect on T. cruzi replication. In contrast, 54, 73, or 30% inhibition of T. gondii replication was observed in the cells treated with rIFN-γ alone, rIFN-γ plus rTNF-α, or TNF-α alone, respectively. The replication of T. gondii tachyzoites in cytokine-activated cells was restored by the addition of extra tryptophan to the culture medium. Similarly,T. gondii tachyzoites transfected with bacterial tryptophan synthase were not sensitive to the microbiostatic effect of rIFN-γ. We also investigated the basis of the cytokine effect on parasite replication by using the three mutant cell lines B3, B9, and B10 derived from 2C4 and expressing defective STAT1α (signal transducer and activator of transcription), JAK2 (Janus family of cytoplasmic tyrosine kinases), or JAK1, respectively, three important elements of a signaling pathway triggered by rIFN-γ. We found that rTNF-α was able to induce low levels expression of INDO mRNA in the parental cell line, as well as the cell line lacking functional JAK2. In contrast to the parental cell line (2C4), rIFN-γ was not able to induce the expression of INDO mRNA or microbiostatic activity in any of the mutant cell lines. These findings indicate the essential requirement of the JAK/STAT pathway for the induction of high levels of INDO mRNA, tryptophan degradation, and the anti-Toxoplasma activity inside human nonprofessional phagocytic cells.

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