RRM1 Antibody

What is RRM1 and what is its primary function in cellular biology?

RRM1 is the catalytic subunit of ribonucleotide reductase, an enzyme that plays a pivotal role in converting ribonucleotides (NTP) into deoxyribonucleotides (dNTP), which are essential for DNA replication and repair . Beyond this enzymatic function, RRM1 has non-reductase roles in DNA damage repair pathways, particularly in homologous recombination (HR) repair. Its expression is independent of the cell cycle, suggesting functions beyond simply providing raw materials for DNA synthesis .

What is the typical structural composition of RRM1?

RRM1 comprises three structural domains: a helical N-terminal domain, an α/β parallel domain, and an αβααβ domain. The active site is located between the N-terminus and C-terminus . The C-terminal region (amino acids 731-793) is particularly important as it facilitates binding with USP11 and recruitment to LaminB1 after irradiation, which is critical for promoting HR and enhancing cell radiation resistance .

What are the primary methods for detecting RRM1 expression in tissue samples?

Two primary methods are used to detect RRM1 expression:

• Immunohistochemistry (IHC): A well-established, convenient, and cost-effective technique for detecting RRM1 protein expression directly in tissue samples .

• Reverse Transcriptase PCR (RT-PCR): Currently the most sensitive method for detecting and quantifying RRM1 mRNA expression .

According to meta-analysis data, these methods have different detection rates: IHC shows approximately 43.8% positive expression while RT-PCR detects 55.5% positive expression across studies .

Research shows significant difference between the two methods in detection rate (χ² = 43.46, P = 0.000) .

What are the recommended applications for RRM1 antibodies in laboratory research?

RRM1 antibodies have been validated for multiple research applications:

• Western Blotting: Typically using a 1:1,000 dilution to detect RRM1 in cell lysates (e.g., HeLa, A549, COS-7) .

• Flow Cytometry: Approximately 0.1 μg can detect RRM1 in one million cells .

• Immunohistochemistry on Paraffin-embedded Tissues: A 1:100 dilution typically works for human tissue sections .

• Affinity Binding Assays: Can be used to determine binding properties of the antibody to RRM1 .

The optimal working dilutions should be determined experimentally as they may vary depending on specimen type and experimental conditions .

How does RRM1 expression influence cancer prognosis?

High RRM1 expression has been associated with poor prognosis in multiple cancer types. For instance:

• In gastric cancer, high RRM1 expression is associated with lymph node involvement, tumor size, Ki67 expression, histological subtype, and histological grade (p<0.05) . Kaplan-Meier analysis demonstrated that high RRM1 expression predicted poor survival in gastric cancer patients .

• In multiple myeloma, patients with higher RRM1 expression have shortened survival . This is supported by in vitro experiments showing that knockdown of RRM1 triggered significant growth inhibition and apoptosis in multiple myeloma cells .

• In advanced nasopharyngeal carcinoma (NPC), RRM1 expression has been linked to progression-free survival (PFS). Patients with RRM1-negative tumors had a median PFS of 7 months compared to 5 months in RRM1-positive cases (p=0.036) .

What is the relationship between RRM1 expression and gemcitabine resistance?

Multiple studies have demonstrated that high RRM1 expression is associated with resistance to gemcitabine-based chemotherapy :

These findings suggest that RRM1 testing could potentially guide treatment selection, though large-scale prospective studies are still needed to further validate this approach .

What molecular mechanisms explain RRM1's role in DNA damage repair?

Recent research has uncovered novel mechanisms by which RRM1 contributes to DNA damage repair:

• RRM1 promotes homologous recombination (HR) by upregulating the expression of RAD51AP1, a critical HR factor, in an E2F1-dependent manner .

• RRM1 interacts with USP11 in the cytoplasm, and upon ionizing radiation (IR), RRM1 is recruited to LaminB1, facilitating USP11 binding to the nuclear pore complex, thus promoting USP11 entry into the nucleus .

• Once in the nucleus, USP11 binds to E2F1 and inhibits its ubiquitin-mediated degradation, thereby enhancing the transcriptional expression of RAD51AP1 .

• Knockdown of RRM1 leads to upregulation of DNA damage response genes, including γ-H2A.X, ATM, ATR, Chk1, Chk2, RAD51, 53BP1, BRCA1, and BRCA2 .

• In p53 wild-type cells, RRM1 knockdown activates p53, p21, Noxa, and Puma, triggering apoptotic cell death .

These findings suggest that RRM1 functions beyond its enzymatic role, playing a critical part in DNA damage repair signaling pathways.

How can experimental design address the dual enzymatic and non-enzymatic functions of RRM1?

To distinguish between RRM1's enzymatic and non-enzymatic functions, researchers can implement several experimental approaches:

• Mutational Analysis: Use of specific RRM1 mutants, such as those lacking amino acids 731-793 (critical for interaction with USP11), can reveal non-enzymatic functions while preserving or disrupting the catalytic domain .

• Domain-specific Targeting: Comparing the effects of truncated RRM1 (e.g., 1-730 amino acids) with full-length RRM1 on DNA repair efficiency, cell survival, and protein interactions .

• Enzymatic Activity Assessment: Measuring dNTP pool changes following RRM1 knockdown or inhibition to assess its enzymatic function .

• Compartmentalization Studies: Investigating RRM1 localization (nuclear vs. cytoplasmic) following DNA damage induction to understand subcellular dynamics and interactions .

• Pathway Analysis: Combining RRM1 manipulation with inhibitors of specific DNA repair pathways (e.g., PARP inhibitors) to determine functional relationships. For example, RRM1 knockdown cells display increased sensitivity to Olaparib following ionizing radiation, supporting RRM1's role in promoting HR .

What are the methodological considerations for using RRM1 antibodies in prognostic studies?

When using RRM1 antibodies for prognostic studies, researchers should consider:

• Method Selection: Determine whether IHC or RT-PCR is more appropriate based on study goals. Consider that detection rates differ significantly between methods (43.8% vs. 55.5%) .

• Standardization: Establish standardized scoring systems for RRM1 positivity to ensure consistency across studies and facilitate meta-analyses .

• Sample Collection and Processing: Standardize tissue fixation, processing, and storage to minimize preanalytical variables that might affect RRM1 detection .

• Validation: Consider using both protein and mRNA detection methods in a subset of samples to validate findings, as some studies have found correlation between mRNA and protein expression levels .

• Controls: Include appropriate positive and negative controls, and consider using cell lines with known RRM1 expression levels as reference standards .

• Clinical Context: Combine RRM1 expression data with comprehensive clinical information, as RRM1's prognostic value may vary depending on treatment regimens, cancer subtypes, and patient characteristics .

How might RRM1 serve as a therapeutic target in cancer treatment?

Evidence suggests RRM1 could be a valuable therapeutic target:

• In multiple myeloma, RRM1 knockdown triggered significant growth inhibition and apoptosis, even in the presence of bone marrow microenvironment .

• The non-specific RRM1 inhibitor clofarabine (CLO) has shown promise in inducing growth arrest and apoptosis in p53 wild-type cancer cell lines .

• Combining RRM1 inhibition with DNA-damaging agents like melphalan has demonstrated synergistic toxicity in experimental models .

• In vivo studies have shown significantly reduced tumor growth in RRM1-knockdown multiple myeloma cells in a murine human MM cell xenograft model .

• The C-terminus of RRM1 (amino acids 731-793) has been identified as particularly important for its non-enzymatic functions in promoting HR and enhancing cell radiation resistance, suggesting potential for targeted therapeutic approaches .

These findings provide rationale for developing more specific RRM1 inhibitors and combining them with existing chemotherapeutic agents to improve patient outcomes.

What advanced experimental approaches can detect changes in RRM1 activity following drug treatment?

To assess changes in RRM1 activity after drug treatment, researchers can employ:

• dNTP Pool Analysis: Measure cellular dNTP levels using sensitive liquid chromatography-mass spectrometry (LC-MS) methods to directly assess RRM1's enzymatic function .

• Protein-Protein Interaction Studies: Use co-immunoprecipitation or proximity ligation assays to monitor RRM1's interactions with binding partners like USP11 and LaminB1 following treatment .

• Subcellular Fractionation: Assess RRM1 distribution between cytoplasmic and nuclear compartments using antibodies specific to RRM1, as translocation patterns may indicate functional changes .

• DNA Damage Assessment: Monitor γ-H2AX foci formation, RAD51 foci, or 53BP1 localization as indicators of DNA damage response activity related to RRM1 function .

• Reporter Systems: Utilize DR-GFP and EJ5-GFP plasmid reporting systems to specifically measure HR and NHEJ repair activity in the context of RRM1 modulation .

These approaches provide complementary information about both enzymatic and non-enzymatic functions of RRM1 following therapeutic intervention.