Journal: Clinical Hematology International

Article Title: The Clinical and Prognostic Significance of Ribonucleotide Reductase Subunits RRM1 and RRM2 mRNA Levels in Patients with Chronic Lymphocytic Leukemia

doi: 10.1007/s44228-023-00033-x

Figure Lengend Snippet: Patient characteristics

Article Snippet: The following assays were used: RRM1:Hs01040698_m1, RRM2: Hs00357247_g1 and GAPDH: Hs02786624_g1 (Applied Biosystems, Waltham, MA, USA).

Techniques: Biomarker Discovery, Mutagenesis, Expressing, Western Blot, Methylation

Journal: Clinical Hematology International

Article Title: The Clinical and Prognostic Significance of Ribonucleotide Reductase Subunits RRM1 and RRM2 mRNA Levels in Patients with Chronic Lymphocytic Leukemia

doi: 10.1007/s44228-023-00033-x

Figure Lengend Snippet: RRM2 mRNA expression and patient parameters

Article Snippet: The following assays were used: RRM1:Hs01040698_m1, RRM2: Hs00357247_g1 and GAPDH: Hs02786624_g1 (Applied Biosystems, Waltham, MA, USA).

Techniques: Expressing

Journal: Clinical Hematology International

Article Title: The Clinical and Prognostic Significance of Ribonucleotide Reductase Subunits RRM1 and RRM2 mRNA Levels in Patients with Chronic Lymphocytic Leukemia

doi: 10.1007/s44228-023-00033-x

Figure Lengend Snippet: Correlation of RRM1 gene promoter methylation status and RRM1/RRM2 mRNA expression

Article Snippet: The following assays were used: RRM1:Hs01040698_m1, RRM2: Hs00357247_g1 and GAPDH: Hs02786624_g1 (Applied Biosystems, Waltham, MA, USA).

Techniques: Methylation, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance 1 2 3

doi: 10.1016/j.neo.2014.05.007

Figure Lengend Snippet: Pyrimidine metabolism–associated RRM2 is differently expressed in breast cancer subtypes and is a predictor of outcome. (A) Box plot shows the relative expression of RRM2 in basal and luminal BCa tissues. (B) KM plot showing the association of RRM2 expression with time to metastasis-free survival in patients with BCa ( N = 1340). Higher expression of RRM2 was significantly (log-rank P = 3.6E-09) associated with poor survival in BCa. (C) Table shows results of univariate Cox P values of RRM2 in each of the publically available data sets and its association with distant metastasis-free survival. (D) KM plot shows the association of RRM2 expression with time to metastasis-free survival in patients with tamoxifen-treated BCa (Loi data set, N = 149). Higher expression of RRM2 was significantly (log-rank P = .002) associated with tumors having intrinsic tamoxifen resistance and poor survival in this patient group. (E) RRM2 protein expression was generally higher in patients who did not respond to TAM compared to those who responded to the treatment. (F) RRM2 expression was significantly higher (Wilcoxon rank sum, P = .04) in patients who were reported to be dead versus those who were alive, post-TAM treatment for a median follow-up time of 8 years. G) KM plot confirms significant association (log-rank test, P = .04) of RRM2 expression with early tumor recurrence.

Article Snippet: Slides were loaded on a Dako (X0909, Dako, CA) autostainer, serum-free protein block (Dako No. X0909) was applied for 5 minutes and blown off, and the RRM2 antibody (SC-81850, Santa Cruz Biotechnology (Santa Cruz (Dallas, TX)) was applied at 1:500 dilution for 1 hour.

Techniques: Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Pathway-Centric Integrative Analysis Identifies RRM2 as a Prognostic Marker in Breast Cancer Associated with Poor Survival and Tamoxifen Resistance 1 2 3

doi: 10.1016/j.neo.2014.05.007

Figure Lengend Snippet: Elevated expression of RRM2 is associated with acquired tamoxifen resistance in BCa and could be targeted to sensitize the tumors to tamoxifen treatment. (A–D) Box plot shows the relative ratios of product:substrate for RRM2, i.e., dUDP/UDP and dCDP/CDP, in TAM-R ( n = 3 biologic replicates) and TAM-S (each n = 3 biologic replicates) cell lines (A and B) and xenograft tissues (C and D), (TAM-R, n = 3 biologic replicates; TAM-S, n = 2 biologic, each in n = 2 technical replicates). The relative ratios for the product:substrates of RRM2 were higher in tamoxifen-resistant (TAM-R) cells ( P = .04 and .19) and tissues ( P = .002 and .001), compared to their parental counterparts. (E) Immunoblot analysis shows levels of RRM2 protein expression in TAM-R and TAM-S cells. β-Actin was used as a loading control. (F) Transcript levels of RRM1, RRM2, and RRM2B relative to 18S RNA in TAM-R and TAM-S cells are presented. (G) Immunoblot analysis to verify RRM2 KD in TAM-R cells. β-actin was used as a loading control. (H) RRM2 KD in TAM-R cells resulted in a significant decrease in proliferation (rank sum P = .0001) compared to control siRNA-treated cells, as assessed by BrdU assay. (I) Transcript levels of CCND2, CCNE1, CCNA1, and CCNB1 were measured after 48 hours of post-RRM2 siRNA or Ctrl siRNA treated cells. (J) Immunoblot analysis of aza-treated TAM R cells shows reduction in RRM2 expression. (K) TAM-R cells were treated with increasing concentrations of aza, and after 72 hours, an MTT-based assay was performed to determine the cell growth and survival.

Article Snippet: Slides were loaded on a Dako (X0909, Dako, CA) autostainer, serum-free protein block (Dako No. X0909) was applied for 5 minutes and blown off, and the RRM2 antibody (SC-81850, Santa Cruz Biotechnology (Santa Cruz (Dallas, TX)) was applied at 1:500 dilution for 1 hour.

Techniques: Expressing, Western Blot, Control, BrdU Staining, MTT Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, RRM2 mRNA expression in pancreatic cancer cell lines relative to expression identified in HPDE. Columns, mean of triplicate; bars, SD. n = 3. B, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDa) in whole cell lysates of HPDE and pancreatic cancer cell lines. C, Expression of let- 7 family members in pancreatic cancer cell lines relative to expression in HPDE. Columns , mean of triplicate; bars , SD. n = 3; * P <0.05. D, RRM2 is a direct target of let-7 . 293TA and MIA PaCa-2 cells were virally infected for expression of precursors of let-7a-1 , let-7a-2 , let-7a-3 , let-7b , and miR-214 ( negative control ) and subsequently transfected with a RRM2 3′ UTR luciferase reporter construct. Luciferase activities measured 36 h after transfection (normalized relative to renilla activity) were plotted. Columns , mean of triplicate; bars , SD. n = 3. * p <0.05, ** p <0.01.

Article Snippet: TaqMan primers and probes for RRM2 (Hs00357251_g1) were obtained from Applied Biosystems (Foster City, CA).

Techniques: Expressing, Western Blot, Infection, Negative Control, Transfection, Luciferase, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A , Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) in whole cell lysates of MIA PaCa-2 overexpressing precursors of let-7 family members. Ratios of RRM2 to β-actin band intensities (normalized to control) from three experiments are indicated ( top ). Asterisks indicate significant reductions ( p <0.05) in RRM2 levels compared with control. B , Immunocytochemical detection of RRM2 in exponentially growing MIA PaCa-2 overexpressing pre- let-7 family members. Original magnification, x20. C , MIA PaCa-2 cells stably overexpressing pre- let-7 family members ( red ) or vector alone ( blue ) were treated with gemcitabine (0.1 nM to 100 µM), and percent inhibition of cellular proliferation was measured using an MTT assay. Points , mean of triplicate; bars , SE. n = 3. Gemcitabine IC 50 estimations indicated ( parentheses ).

Article Snippet: TaqMan primers and probes for RRM2 (Hs00357251_g1) were obtained from Applied Biosystems (Foster City, CA).

Techniques: Western Blot, Control, Stable Transfection, Plasmid Preparation, Inhibition, MTT Assay

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A, Western blotting analysis of hENT1 (∼50–55 kDa), hENT2 (50 kDa), hCNT1 (72 kDa), hCNT3 (77 kDa), CDA (55 kDa), dCK (30 kDa), RRM1 (94 kDa), RRM2 (45 kDa), and β-actin (45 kDA) levels in whole cell lysates of Capan-1 and Capan-1-GR cells. B , RRM2 protein increased in a gemcitabine dose-dependent fashion in Capan-1-GR cells. C, Western blotting analysis of RRM2 (∼45 kDa) and β-actin (45 kDA) levels in cells with acquired gemcitabine resistance. Ratios of RRM2 to β-actin band intensities (normalized to untreated cells) from three experiments are indicated ( top ). Asterisk indicates significantly higher RRM2 expression in gemcitabine-resistant cells ( p <0.05) compared with untreated cells. D and E, Relative expression of precursor and mature let-7a in Capan-1 ( D ) and L3.6pl ( E ) cells induced to acquire gemcitabine resistance. Columns, mean of triplicate; bars, SD. n = 3. F , Differential miRNA expression in Capan-1-GR compared with Capan-1. Putative RRM2-modulating miRNAs and their extent of reduction in Capan-1-GR cells are shown ( right ).

Article Snippet: TaqMan primers and probes for RRM2 (Hs00357251_g1) were obtained from Applied Biosystems (Foster City, CA).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Differential Processing of let-7 a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

doi: 10.1371/journal.pone.0053436

Figure Lengend Snippet: A and B, Relative expression of primary let-7a transcripts ( A ) and mature let-7a ( B ) in 2 normal pancreatic tissues and 10 PDAC samples representing various tumor stages. C , Ratios of mature to precursor let-7a transcripts calculated from data points in A and B . D, Western blotting analysis of RRM2 (45 kDa) in total lysates (50 µg) of 6 matched normal-PDAC pairs. Ratios of RRM2 band intensities in PDACs compared to matched normal tissues indicated ( top ). Asterisk indicates significantly higher RRM2 expression in PDAC tissues ( p <0.05) compared with matched normal tissues. E and F, Relative expression of primary let-7a transcripts ( E ) and mature let-7a ( F ) in matched normal-PDAC pairs. G , Ratios of mature to precursor let-7a transcripts calculated from data points in E and F . Asterisk indicates significantly lower mature to precursor let-7a ratio in PDAC tissues ( p <0.05) compared with matched normal tissues.

Article Snippet: TaqMan primers and probes for RRM2 (Hs00357251_g1) were obtained from Applied Biosystems (Foster City, CA).

Techniques: Expressing, Western Blot

Journal: Leukemia

Article Title: Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine

doi: 10.1038/leu.2013.330

Figure Lengend Snippet: Expression of genes implicated in the metabolism of AZA. ( a ) Expression of UCK1 , UCK2 , DCK , hENT1 , RRMI and RRM2 (relative to GAPDH ) in patients with MDS ( n =57) before AZA treatment. ( b ) Expression of UCK1 (relative to GAPDH ) in patients without response to AZA (SD or PD) vs patients with response to AZA (CR, PR, HI and mCR). In both plots, the thick middle line represents the median expression of the different genes, and the top and bottom edges of the box represent the 75th and 25th percentiles, respectively. The outliers are also represented by open and filled circles and asterisks.

Article Snippet: Specific oligonucleotides and TaqMan probes were available from Applied Biosystems (catalog no: UCK1: Hs01075618_m1; UCK2 : Hs:00367072_m1, DCK : Hs00176127_m1, hENT1 : Hs01085706_m, RRM1 : Hs01040698_m1, RRM2 : Hs01072069_g1).

Techniques: Expressing

Journal: Leukemia

Article Title: Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine

doi: 10.1038/leu.2013.330

Figure Lengend Snippet: Molecular alterations of UCK1 in MDS patients. ( a ) Representative examples of methylation-specific PCR analysis of UCK1 in seven MDS cases without response to AZA and six cases of MDS patients with response to AZA. C(+), DNA universally methylated; C(−), healthy individual; blank, control without added DNA; U, unmethylated alleles; and M, methylated. ( b ) UCK1 gene locus and the sequenced regions. Five PCR were designed to sequence the complete coding DNA sequence (Exon 1, Exon 2, Exon 3-5, Exon 6 and Exon 7) for all the isoforms of the UCK1 protein. A total of seven common genetic variants, indicated by their reference number, were found to be polymorphic in at least one subject. ( c ) UCK1 expression levels in different genotype groups. Gene expression values were compared with the genotypes of the genetic variants identified. In the plots, the thick middle line represents the median expression of the different genes, and the top and bottom edges of the box represent the 75th and 25th percentiles, respectively. The outliers are represented by asterisks.

Article Snippet: Specific oligonucleotides and TaqMan probes were available from Applied Biosystems (catalog no: UCK1: Hs01075618_m1; UCK2 : Hs:00367072_m1, DCK : Hs00176127_m1, hENT1 : Hs01085706_m, RRM1 : Hs01040698_m1, RRM2 : Hs01072069_g1).

Techniques: Methylation, Control, Sequencing, Expressing, Gene Expression

Journal: Leukemia

Article Title: Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine

doi: 10.1038/leu.2013.330

Figure Lengend Snippet: Silencing of the UCK1 gene. Effect of siUCK1-1 on the expression of the UCK1 gene (relative to GAPDH ) was evaluated using quantitative PCR ( a ) and western blotting analysis ( b ). Downregulation of the UCK1 mRNA by siUCK1-1 was observed in non-transfected compared with mock transfected control cells, both not treated (* P =0.046) and treated with AZA (** P =0.021) ( a ). Mean of two different experiments is shown. UCK1 protein expression was equally diminished in K562 cells exposed to siUCK1-1 ( b ). ( c ) Apoptosis (Annexin V-FITC (fluorescein isothiocyanate) and PI staining) was measured in K562 cells cultured with SRC, TKO and siUCK1-1, without AZA and with 24-h AZA. AZA induced an increase in Annexin V-positive cells, but apoptosis was blunted in K562 cells when siUCK1-1 was present and UCK1 , as well as UCK1 protein, were downexpressed (* P =0.0015, siUCK1-1 transfected vs control culture).

Article Snippet: Specific oligonucleotides and TaqMan probes were available from Applied Biosystems (catalog no: UCK1: Hs01075618_m1; UCK2 : Hs:00367072_m1, DCK : Hs00176127_m1, hENT1 : Hs01085706_m, RRM1 : Hs01040698_m1, RRM2 : Hs01072069_g1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Staining, Cell Culture

Journal: Leukemia

Article Title: Expression of nucleoside-metabolizing enzymes in myelodysplastic syndromes and modulation of response to azacitidine

doi: 10.1038/leu.2013.330

Figure Lengend Snippet: OS by UCK1 expression. Kaplan–Meier analysis. Solid lines indicate OS of patients with expression of UCK1 above the median; dashed lines indicate OS of patients with expression of UCK1 below the median.

Article Snippet: Specific oligonucleotides and TaqMan probes were available from Applied Biosystems (catalog no: UCK1: Hs01075618_m1; UCK2 : Hs:00367072_m1, DCK : Hs00176127_m1, hENT1 : Hs01085706_m, RRM1 : Hs01040698_m1, RRM2 : Hs01072069_g1).

Techniques: Expressing