Dihydropyrimidine Dehydrogenase Levels in Colorectal Cancer Cells Treated with a Combination of Heat Shock Protein 90 Inhibitor and Oxaliplatin or Capecitabine

Purpose: Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme in the catabolism of fluoropyrimidine drugs including capecitabine. A recent report has suggested that oxaliplatin chemotherapy is associated with elevated DPD levels and chemoresistance pattern. As a newly developed chemotherapeutic agent, 17-allyloamino-17-demethoxy-geldanamycin (17-AAG) can be effective in combination therapy with oxaliplatin and capecitabine in colorectal cancer (CRC). DPD expression level can be a predictive factor in oxaliplatin and capecitabine-based chemotherapy. We evaluated DPD in mRNA and protein levels with new treatments: 17-AAG in combination with oxaliplatin and capecitabine in HT-29 and HCT-116 cell lines. Methods: Drug sensitivity was determined by the water-soluble tetrazolium-1 assay in a previous survey. Then, we evaluated the expression levels of DPD and its relationship with the chemotherapy response in capecitabine, oxaliplatin, and 17-AAG treated cases in single and combination cases in two panels of CRC cell lines. DPD gene and protein expression levels were determined by real-time polymerase chain reaction and western blotting assay, respectively. Results: DPD gene expression levels insignificantly increased in single-treated cases versus untreated controls in both cell lines versus controls. Then, the capecitabine and oxaliplatin were added in double combinations, where DPD gene and protein expression increased in combination cases compared to pre-chemotherapy and single drug treatments. Conclusion: The elevated levels of cytotoxicity in more effective combinations could be related to a different mechanism apart from DPD mediating effects or high DPD level in the remaining resistance cells (drug-insensitive cells), which should be investigated in subsequent studies.

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Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2796.2796 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2796-2796

Author(s):

Christof Schneider ◽

Dirk Winkler ◽

Meike Loddenkemper ◽

Alexander Krober ◽

Peter Lichter ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cyclin D1 ◽

Candidate Genes ◽

Cell Lines ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Expression Levels ◽

Protein Levels ◽

Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

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Correlations of Clinicopathological Factors with Protein Expression Levels of Thymidylate Synthase, Dihydropyrimidine Dehydrogenase and Orotate Phosphoribosyltransferase in Colorectal Cancer

Chemotherapy ◽

10.1159/000313524 ◽

2010 ◽

Vol 56 (2) ◽

pp. 120-126 ◽

Cited By ~ 4

Author(s):

Tetsuo Sumi ◽

Kenji Katsumata ◽

Akihiko Tsuchida ◽

Tetsuo Ishizaki ◽

Motohide Shimazu ◽

...

Keyword(s):

Colorectal Cancer ◽

Protein Expression ◽

Thymidylate Synthase ◽

Dihydropyrimidine Dehydrogenase ◽

Clinicopathological Factors ◽

Orotate Phosphoribosyltransferase ◽

Expression Levels

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Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Blood ◽

10.1182/blood.v124.21.5957.5957 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5957-5957

Author(s):

Marie-Magdelaine Coudé ◽

Thorsten Braun ◽

Jeannig Berrou ◽

Mélanie Dupont ◽

Raphael Itzykson ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Mrna Expression ◽

Rna Polymerase Ii ◽

Cell Lines ◽

Leukemia Cell ◽

K562 Cells ◽

Antileukemic Activity ◽

Protein Levels ◽

Gene And Protein Expression

Abstract Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

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High Expression of LTBP2 Contributes to Poor Prognosis in Colorectal Cancer Patients and Correlates with the Mesenchymal Colorectal Cancer Subtype

Disease Markers ◽

10.1155/2019/5231269 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Ying Huang ◽

Guihua Wang ◽

Chunmei Zhao ◽

Rong Geng ◽

Shu Zhang ◽

...

Keyword(s):

Colorectal Cancer ◽

Overall Survival ◽

Protein Expression ◽

Mrna Expression ◽

Cell Lines ◽

Critical Role ◽

Prognostic Significance ◽

Poor Overall Survival ◽

Expression Levels ◽

Mrna And Protein Expression

Colorectal cancer (CRC) is a complex and heterogeneous disease with four consensus molecular subtypes (CMS1-4). LTBP2 is a member of the fibrillin/LTBP super family and plays a critical role in tumorigenesis by activating TGF-β in the CMS4 CRC subtype. So far, the expression and prognostic significance of LTBP2 in CRC remains obscure. In this study, we aimed to analyze the mRNA and protein expression levels of LTBP2 in CRC tissues and then estimate their values as a potential prognostic biomarker. We detected the mRNA expression of LTBP2 in 28 cases of fresh CRC tissues and 4 CRC cell lines and the protein expression of LTBP2 in 483 samples of CRC tissues, matched tumor-adjacent tissues, and benign colorectal diseases. LTBP2 protein expression was then correlated to patients’ clinical features and overall survival. Both LTBP2 mRNA and protein expression levels in CRC tissues were remarkably superior to those in adjacent normal colorectal tissues (P=0.0071 and P<0.001, respectively), according to TCGA dataset of CRC. High LTBP2 protein expression was correlated with TNM stage (P<0.001), T stage (P<0.001), N stage (P<0.001), and M stage (P<0.001). High LTBP2 protein expression was related to poor overall survival in CRC patients and was an independent prognostic factor for CRC. LTBP2 mRNA expression was especially higher in the CMS4 subtype (P<0.001), which was confirmed in CRC cell lines. Our data suggested that LTBP2 may act as an oncogene in the development of colorectal cancer and have important significance in predicting CRC prognosis. LTBP2 could be a novel biomarker and potential therapeutic target for mesenchymal colorectal cancer and can improve the outcome of high-risk CRC.

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Increased Musashi-2 and Decreased NUMB Protein Levels Observed in Human Colorectal Cancer are reverted to Normal Levels by ATRA-Induced Cell Differentiation

International Journal of Cancer Research & Therapy ◽

10.33140/ijcrt/03/02/00003 ◽

2018 ◽

Vol 3 (2) ◽

Keyword(s):

Colorectal Cancer ◽

Protein Expression ◽

Cell Lines ◽

Cellular Differentiation ◽

Tissue Samples ◽

Colorectal Tumorigenesis ◽

Protein Levels ◽

Colon Tumorigenesis ◽

Epithelial Homeostasis ◽

All Trans Retinoic Acid

Background: Musashi stem cell (SC) proteins (MSI-1 & MSI-2) are known to become over expressed during colorectal tumorigenesis in humans and mice. MSI-1 overexpression induces tumorigenesis through Notch activation via inactivation of NUMB. Previous studies also show that MSI-2 overexpression in mice induces intestinal tumorigenesis but the mechanism is independent of NUMB. However, whether the MSI-2/NUMB pathway contributes to colorectal cancer (CRC) development in humans is still undetermined. Methods: We evaluated expression of MSI-2 and NUMB proteins in matched normal and CRC patient samples, as well as in human CRC cell lines. We also determined whether induction of cellular differentiation by all-trans retinoic acid (ATRA) influences MSI-2 and NUMB expression. Results: Analysis of matched patient tissue samples and CRC cell lines showed that MSI-2 protein expression is significantly increased and NUMB expression is decreased in CRCs compared to the normal colonic tissue. Immunostaining of normal and adenomatous colonic epithelium revealed that MSI-1+ and MSI-2+ SCs reside in the SC niche and they become overpopulated during colon tumorigenesis. Moreover, promoting cellular differentiation by ATRA reduces MSI-2 protein levels, while increasing NUMB protein levels in human CRC cell lines. Conclusions: MSI-2/NUMB protein expression is altered during colon tumorigenesis, and indicates that MSI-2/ NUMB signaling in human colonic stem cells is closely linked to normal colonic epithelial homeostasis. Implications: The ability to normalize MSI-2/NUMB signaling by inducing differentiation of cancer SCs suggests a novel therapeutic approach for CRC treatment.

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Patient-derived gene and protein expression signatures of NGLY1 deficiency

The Journal of Biochemistry ◽

10.1093/jb/mvab131 ◽

2021 ◽

Author(s):

Benedikt Rauscher ◽

William F Mueller ◽

Sandra Clauder-Münster ◽

Petra Jakob ◽

M Saiful Islam ◽

...

Keyword(s):

Protein Expression ◽

Cell Lines ◽

Web Application ◽

Ribosome Biogenesis ◽

Genetic Disorder ◽

Cell Types ◽

Cell Type ◽

Protein Levels ◽

Gene And Protein Expression ◽

Complex Genetic Disorder

Abstract N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

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Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models

Gastroenterology Research and Practice ◽

10.1155/2020/1093235 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Layanne C. C. Araujo ◽

Silvana Bordin ◽

Carla R. O. Carvalho

Keyword(s):

Protein Expression ◽

Reference Gene ◽

Reference Genes ◽

Lipid Droplets ◽

Standard Diet ◽

Experimental Conditions ◽

Nonalcoholic Fatty Liver ◽

Expression Levels ◽

Protein Levels ◽

Gene And Protein Expression

The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of β-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

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Patient-derived gene and protein expression signatures of NGLY1 deficiency

10.1101/2021.07.28.453930 ◽

2021 ◽

Author(s):

Benedikt Rauscher ◽

William F. Mueller ◽

Sandra Clauder-Muenster ◽

Petra Jakob ◽

M. Saiful Islam ◽

...

Keyword(s):

Protein Expression ◽

Cell Lines ◽

Web Application ◽

Genetic Disorder ◽

Cell Types ◽

Cell Type ◽

Protein Levels ◽

Gene And Protein Expression ◽

Proteasomal Genes ◽

Complex Genetic Disorder

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient- derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

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