scholarly journals 90 Establishment and characterization of Day 30 equine chorionic girdle and allantochorion cell lines

2020 ◽  
Vol 32 (2) ◽  
pp. 171
Author(s):  
S. Salman ◽  
A. Asghar ◽  
C. Magee ◽  
Q. Winger ◽  
G. Bouma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Vector System ◽  
Stable Cell Line ◽  
Rt Pcr ◽  
Study Gene Expression ◽  
Lentiviral Infection ◽  
The Media ◽  
Equine Chorionic Gonadotropin

Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production invitro from Day 30 CG and AC cells. Three mares (n=3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured invitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7±2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42ngmL−1) invitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042ngmL−1) invitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced invitro as early as Day 30 from CG cells.

scholarly journals Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

2020 ◽  
Author(s):  
Kwan-Sik Min ◽  
Jong-Ju Park ◽  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Myung-Hwa Kang
Keyword(s):  
Gene Expression ◽  
Chorionic Gonadotropin ◽  
Expression Profiles ◽  
Expression System ◽  
Laboratory Animals ◽  
Metabolic Clearance ◽  
Qrt Pcr ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

Comprehensive Analysis of Homeobox Gene Expression in Hodgkin Lymphoma Cell Lines Reveals Similarities to Prostate Carcinoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 966-966
Author(s):  
Stefan Nagel ◽  
Christof Burek ◽  
Hilmar Quentmeier ◽  
Corinna Meyer ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Prostate Carcinoma ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Cell Cycle Regulators ◽  
Rt Pcr

Abstract Homeobox genes code for transcription factors with essential regulatory impact on cellular processes during embryogenesis and in the adult. Increasingly, members of the circa 200 gene strong family are emerging as major oncogenic players, prompting our investigation into possible homeobox gene dysregulation in Hodgkin lymphoma (HL) in which no recurrent oncogene involvement has been known. Accordingly, we screened 6 well characterized HL cell lines (HDLM-2, KM-H2, L-1236, L-428, L-540, SUP-HD1) and 3 non-Hodgkin lymphoma (NHL) cell lines (RC-K8, RI-1, SC-1) for homeobox gene expression using Affymetrix U133-2.0 whole-genome oligonucleotide microarrays. Of 15 candidate genes thus shown to reveal HL-specific expression patterns, 5 homeobox genes were shortlisted as potentially key dysregulatory targets in HL after additional RT-PCR expression analysis relative to controls. While 3/5 homeobox genes were upregulated in HL (HOXB9, HOXC8, HLXB9), 2/5 were downregulated (BOB1, PAX5). Furthermore, cloning and sequencing RT-PCR products obtained with degenerate primers recognizing conserved homeobox motifs confirmed the predominant expression of HOXB9 in HL cells. However, fluorescence in situ hybridization (FISH) analysis of the HOXB locus (at 17q21) revealed no cytogenetic aberrations, indicating that its activation is conducted non-chromosomally in HL cells. Surprisingly, known target genes of HOXB9 and HOXC8 remained unperturbed, implying novel downstream effector pathways in HL cells. Antisense oligos directed against HOXB9 and forced expression experiments using cloned full length HOXB9 cDNA indicated its involvement in both proliferation and apoptosis. Cell cycle regulators BTG1, BTG2 and GEMININ have been described to interact with HOXB9 and may represent potential targets deserving investigation. We recently showed that HLXB9 promotes IL6 expression in HL cells in response to a constitutively active PI3K signalling pathway therein (Nagel et al., Leukemia19, 841–6, 2005). Our most recent data indicate that HLXB9 is also expressed in various NHL cell lines including anaplastic, diffuse and mediastinal large cell as well as follicular B-cell lymphomas while expression is notably absent from Burkitt, mantle cell and natural killer T-cell lymphomas reflecting their pathologic classification. Intriguingly, our data highlight unexpected similarities between HL and prostate cancer cells which together uniquely overexpress HOXB9, HOXC8 and HLXB9 (or its close homolog GBX2). Additional genes expressed in prostate carcinoma (HOXB13, PRAC1, PRAC2) were detected in two HL cell lines (KM-H2 and L-428) suggesting further parallels may be revealed. Detection of downregulated B-cell differentiation factors BOB1 and PAX5 in our panel of HL cell lines validated this approach. Both factors were previously implicated in oncogenesis of HL lacking IGH rearrangements and other key B-cell characteristics. In summary, we identified a unique homeobox gene expression pattern involving HOXB9, HOXB13, HOXC8 and HLXB9 in HL cell lines resembling that of prostate carcinoma cells. Overexpressed HOXB9 contributes to proliferation and protects against apoptosis in HL cells potentially via interacting with cell cycle regulators BTG1/2 and/or GEMININ.

Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3526-3526
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Sophia Adamia ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Genomic Alteration ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

Deletions In TBL1XR1 Results In Glucocorticoid Resistance By Decreasing Glucocorticoid Signaling In Childhood B-Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 602-602
Author(s):  
Courtney L Jones ◽  
Teena Bhatla ◽  
Jinhua Wang ◽  
Wallace Bourgeois ◽  
Bitterman S Danielle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Viability ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Steroid Resistance ◽  
Rt Pcr ◽  
Regulatory Regions ◽  
Prednisolone Treatment ◽  
Gene Regulatory

Abstract Introduction The prognosis for children with acute lymphoblastic leukemia who relapse is poor and discovery of the underlying mechanisms that lead to drug resistance is a top priority. Relapsed blasts have intrinsic chemoresistance compared to diagnosis blasts especially to glucocorticoids (Klumper et. al, 1995). Furthermore, resistance to glucocorticoids is associated with a poor prognosis in childhood ALL (Dördelmann M et. al, Blood 1999, Schmiegelow K et. al, Leukemia 2001, Tissing WJ et. al, Leukemia 2003). We have previously identified recurrent deletions with concordant decreased gene expression in TBL1XR1 in 10.7% of patients at relapse (Hogan et. al, 2011). TBL1XR1 codes for the TBLR1 protein which is responsible for the dismissal and degradation of nuclear corepressor (N-CoR) complex proteins including N-CoR1, SMRT, GPS2, and histone deacetylases (HDAC) (Perissi V et. al, Cell 2004). We hypothesized that TBL1XR1 deletions may result in resistance to glucocorticoid agonist, prednisolone through up-regulation of N-CoR complex proteins. Methods B-precursor ALL cell lines Reh, and RS4;11 were transduced with lentiviral constructs containing control and TBL1XR1 targeting shRNAs. Knockdown was confirmed by RT-PCR and western blotting. Stable cell lines were treated with prednisolone, doxorubicin, 6-thioguanine, or etoposide for 24-48 hours. Cell viability and apoptosis were measured by cell titer glo luminescence assay (promega) and annexin V-PE and 7-Amino-actinomycin D (7AAD) staining (Annexin V-PE Apoptosis Detection Kit, BD Pharmingen, San Diego, CA, USA) respectively. To determine changes in global gene expression by TBL1XR1 knockdown, stable Reh cell lines were treated with prednisolone or vehicle for 8 hours and then collected for RNA extraction (Qiagen, RNeasy mini kit) and microarray analysis. Microarray data was validated by RT-PCR. To elucidate the mechanism of resistance we performed small-scale biochemical fractionation and chromatin immunoprecipitation (ChIP) detecting levels of glucocorticoid receptor (GR), TBLR1, N-CoR1, and HDAC3 residing on the chromatin as well as gene specific glucocorticoid response elements (GREs). Results In this study, we demonstrate that knockdown of TBL1XR1 results in resistance to the glucocorticoid agonist prednisolone but not other classes of chemotherapeutic agents. We discovered that 51 of the 117 genes induced by prednisolone in control cells had decreased induction of at least 50%. We validated a subset of prednisolone induced genes including, GILZ, TXNIP, ZEB1, ST6GALNAC3, IL21R, and CCPG1 by RT-PCR. To explore the mechanism of TBL1XR1 mediated decrease in GR signaling we determined the effect of TBL1XR1 depletion of GR recruitment to total bulk chromatin. In TBL1XR1 knockdown cells, no GR was detected in the chromatin associated fractions in vehicle or prednisolone treatment conditions, despite similar levels of GR protein between control and TBL1XR1 knock down lines. We show that the decreased GR levels is associated with an increased level of NCoR1 detected in the chromatin fraction of TBLR1 depleted cells; however no change in HDAC3 levels were observed. We confirmed these results by interrogating the gene regulatory regions of GILZ and TXNIP by ChIP. In TBL1XR1 depleted lines a decrease in GR occupancy in prednisolone stimulated cells was observed compared to control lines. We also observed increased levels of N-CoR1, and HDAC3 occupying these GREs. To interrogate the functional relationship between increased NCoR1 and HDAC3 levels on the gene regulatory region as a result of TBL1XR1 knockdown we depleted NCoR1 or inhibited HDAC3 using a pan HDAC inhibitor SAHA and examined the impact of prednisolone treatment on cell viability and induction of GILZ. We found that upon NCoR1 depletion or HDAC inhibition, TBL1XR1 knockdown line was no longer resistant to prednisolone and the induction of GILZ was restored. Conclusions Reduction of TBL1XR1 results in prednisolone resistance in ALL by decreasing GR occupancy on gene regulatory regions through the upregulation of the NCoR co-repressor complex at these sites. Our work and others has provided insight into the importance of transcription regulatory complexes in steroid resistance in ALL (and perhaps other malignancies) as well as opportunities for novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2440-2440
Author(s):  
Tian Yuan ◽  
Yaling Yang ◽  
Jeffrey You ◽  
Daniel Lin ◽  
Kefeng Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Rt Pcr ◽  
Altered Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparative gene expression profiling of mouse ovaries upon stimulation with native equine chorionic gonadotropin (eCG) and tethered recombinant-eCG

2020 ◽  
Author(s):  
Kwan-Sik Min ◽  
Jong-Ju Park ◽  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Myung-Hwa Kang
Keyword(s):  
Gene Expression ◽  
Chorionic Gonadotropin ◽  
Expression Profiles ◽  
Expression System ◽  
Laboratory Animals ◽  
Metabolic Clearance ◽  
Qrt Pcr ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin

Abstract Background: Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of native eCG and recombinant eCG (rec-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with native eCG and rec-eCG produced from CHO-suspension (CHO-S) cells. eCG and rec-eCG were cloned and transfected into CHO-S cells and quantified. Thereafter, we determined the metabolic clearance rate (MCR) of native eCG and rec-eCG up to 24 h after intravenous administration through the tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC).Results: Rec-eCG was markedly up-regulated initially after transfection and maintained until recovery on day 9. Oligosaccharide chains were substantially modified in rec-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was slightly lower for rec-eCG than for eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentration, rec-eCG and native eCG were detected in the blood at 24h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by >2-fold in the group receiving rec-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses.Conclusions: The present results indicate that rec-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of rec-eCG with enhanced biological activity in vivo.

scholarly journals The NPM-ALK Oncogenic Kinase Suppresses CGAS-Sting-Associated Anti-Tumor Immune Responses through STAT3 Activation in Anaplastic Large Cell Lymphoma (ALCL)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1338-1338
Author(s):  
Ioanna Xagoraris ◽  
Georgia Kokaraki ◽  
Christina Plastira ◽  
Konstantina Stathopoulou ◽  
Vasiliki Leventaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Responses ◽  
Cell Lines ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Rt Pcr ◽  
Large Cell Lymphoma ◽  
Sting Pathway ◽  
Lymphoma Type

Abstract Background: ALK+ anaplastic large cell lymphoma (ALCL) is a distinct T-cell non-Hodgkin lymphoma type that frequently carries the t(2;5) resulting in overexpression and activation of NPM-ALK chimeric kinase, which activates multiple oncogenic pathways including JAK-STAT3 pathway. The presence of cytosolic DNA of either exogenous or endogenous origin activates the cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, which activates the adaptor protein STING. The latter then activates the TBK1 and IKK kinases, which activate through phosphorylation the transcription factors IRF3 and NF-κB, respectively. IRF3 and NF-κB induce expression of interferons (e.g. IFN-β) and cytokines leading to activation of innate immune responses. The potential role of NPM-ALK oncogenic kinase in cGAS-STING-related anti-tumor immune responses in ALK+ ALCL is unknown to date. Therefore, the present study aimed to investigate the biologic impact of NPM-ALK on cGAS-STING activation status and expression of relevant interferon genes in ALK+ ALCL. Methods: The in vitro system included 5 ALK+ (Karpas 299, SUPM2, DEL, SUDHL1, L82) and 2 ALK- (Mac-1, Mac-2a) ALCL cell lines, as well as Ba/F3 cells stably transfected with NPM-ALK (Ba/F3-NPM-ALK) or a control (Ba/F3-MIG) plasmid. Expression and activation (phosphorylation) of cGAS-STING pathway proteins at baseline and experimental conditions were analysed by RT-PCR and Western blot at the RNA and protein level, respectively. Inhibition of ALK and STAT3 activity was performed using Crizotinib and the selective XIII STAT3 inhibitor, respectively. Silencing of ALK gene was performed using transient transfection with ALK siRNA and the Amaxa Nucleofector Technology. A STING agonist and TBK inhibitor (Amlexanox) were also used alone or in combination with other agents. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of interferon beta (IFN-β), CXCL10, and interferon gamma (IFN-γ), as well as a control gene (GAPDH), with quantitative RT-PCR. The patient study group included 38 previously untreated patients with ALK+ ALCL. Immunohistochemical analysis for STING protein expression was performed using a monoclonal antibody (Cell Signaling) and standard protocols. An arbitrary 10% cutoff was used to define positivity. Results: STING gene was highly expressed at both the mRNA and protein level in ALK+ and ALK- ALCL cell lines, however, cGAS-STING pathway proteins were activated at a variable level among ALCL cell lines as shown in immunoblots. STING was highly expressed in 36 of 38 (95%) ALK+ ALCL tumors, highlighting its biologic significance in this lymphoma type. Inhibition of ALK activity by Crizotinib resulted in significant increase in IFN-β and CXCL10 gene expression linked to activation/phosphorylation of TBK1 indicating cGAS-STING pathway activation in ALK+ ALCL and Ba/F3-NPM-ALK cells. Silencing of ALK gene with specific ALK siRNA also resulted in a dramatic increase in the CXCL10 gene expression (mRNA level). Similarly, treatment of ALK+ ALCL cells with the XIII STAT3 inhibitor resulted in significantly increased IFN-β and CXCL10 gene expression associated with activation of the cGAS-STING pathway proteins in ALK+ ALCL cells but also in the ALK- ALCL cell line, Mac-1. Incubation of ALK+ ALCL cells with a STING agonist alone led to further activation of the cGAS-STING pathway in ALK+ ALCL cells. Conclusion: NPM-ALK suppresses STING-associated, anti-tumor immune responses in ALK+ ALCL, through STAT3 activation and regulation of gene expression of type-1 interferons (IFN-β, CXCL10). Thus, combined ALK inhibition (ALK inhibitors) and STING stimulation (STING agonists) may represent a novel investigational therapeutic strategy for these patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparative gene expression profiling of mouse ovaries upon stimulation with natural equine chorionic gonadotropin (N-eCG) and tethered recombinant-eCG (R-eCG)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kwan-Sik Min ◽  
Jong-Ju Park ◽  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Myung-Hwa Kang
Keyword(s):  
Gene Expression ◽  
Chorionic Gonadotropin ◽  
Expression Profiles ◽  
Expression System ◽  
Laboratory Animals ◽  
Metabolic Clearance ◽  
Qrt Pcr ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin

Abstract Background Equine chorionic gonadotropin (eCG) induces super-ovulation in laboratory animals. Notwithstanding its extensive usage, limited information is available regarding the differences between the in vivo effects of natural eCG (N-eCG) and recombinant eCG (R-eCG). This study aimed to investigate the gene expression profiles of mouse ovaries upon stimulation with N-eCG and R-eCG produced from CHO-suspension (CHO-S) cells. R-eCG gene was constructed and transfected into CHO-S cells and quantified. Subsequently, we determined the metabolic clearance rate (MCR) of N-eCG and R-eCG up to 24 h after intravenous administration through the mice tail vein and identified differentially expressed genes in both ovarian tissues, via quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Results R-eCG was markedly expressed initially after transfection and maintained until recovery on day 9. Glycan chains were substantially modified in R-eCG protein produced from CHO-S cells and eliminated through PNGase F treatment. The MCR was higher for R-eCG than for N-eCG, and no significant difference was observed after 60 min. Notwithstanding their low concentrations, R-eCG and N-eCG were detected in the blood at 24 h post-injection. Microarray analysis of ovarian tissue revealed that 20 of 12,816 genes assessed therein were significantly up-regulated and 43 genes were down-regulated by > 2-fold in the group that received R-eCG (63 [0.49%] differentially regulated genes in total). The microarray results were concurrent with and hence validated by those of RT-PCR, qRT-PCR, and IHC analyses. Conclusions The present results indicate that R-eCG can be adequately produced through a cell-based expression system through post-translational modification of eCG and can induce ovulation in vivo. These results provide novel insights into the molecular mechanisms underlying the up- or down-regulation of specific ovarian genes and the production of R-eCG with enhanced biological activity in vivo.

The MUC16 mucin gene upregulates gene transcripts associated with invasion

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21057-21057
Author(s):  
J. P. Diaz ◽  
S. Kehoe ◽  
D. Thapi ◽  
N. Rosales ◽  
X. Ma ◽  
...  
Keyword(s):  
Growth Rate ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Vector System ◽  
Cell Surface Expression ◽  
Invasion Assay ◽  
Rt Pcr ◽  
Invasive Potential ◽  
Stable Transfectants

21057 Background: The MUC16 gene encodes the CA125 ovarian cancer antigen and may play a role in invasion. The aim of this study is to investigate the properties of MUC16 in vitro. Methods: PCR was used to create a DNA construct, MUC16–1R-GFP, containing all of the MUC 16 sequence proximal to the second cysteine loop repeat and a GFP-vector construct. SKOv-3 and T-80 cell lines were selected because they are negative for immunologically active CA125. Transfections were performed utilizing the phrGFP II C vector system. Stable transfectants were sorted for GFP by FACS and were grown as populations. The growth rate of the transfected populations were compared to vector only. Periodically the stable transfectants were analyzed by FACS for GFP and OC125 mAb. The in vitro invasive potential was examined, using a Matrigel® invasion assay. RT PCR was used to examine the effect of MUC16 constructs for a panel of invasion focused genes. Immunoblotting was performed on the 1R protein and probed for all genes that were significantly changed on RT PCR. Results: The SKOv-3 1R transfected cell lines produce measurable CA125 into the cell culture medium (4–10 U/mL) while T-80 1R cell lines did not. Analysis of the in vitro growth rate did not demonstrate any difference between the transfectant and vector only cell lines. The 1R-GFP transfected lines showed MUC16 cell surface expression by FACS analysis. Invasion assay of the MUC16 transfectants demonstrated substantially more invasive potential than vector only (SKOV-3: 36% vs. 15% invasion; T-80: 35% vs. 23% invasion). Several transcripts associated with invasion and metastasis were up-regulated 3–9 fold including CDH1, FN1, IL1B, MMP7 and MMP9. Transcripts for the tyrosine kinases SYK and HTATIP2 were down-regulated 16–20 fold respectively. Western immunoblotting confirmed the RT PCR findings. Conclusions: The1R-GFP construct did not alter the growth rate of transfectants in vitro; however; the SKOV-3 1R and T-80 1R cell line did have substantially more invasive potential than vector only. Five transcripts associated with invasion were up-regulated, and 2 transcripts for kinases were down-regulated in the transfected cell lines. These data supports an association between MUC 16 and invasive potential. No significant financial relationships to disclose.

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