NDRG1 Human

What is the molecular structure of human NDRG1?

Human NDRG1 is a 394 amino acid protein with a molecular weight of approximately 43 kDa. Its structure includes an NDRG domain (aa 286-316) and three tandem 10 amino acid hydrophilic repeats (aa 339-368). There are three potential isoform variants - two involving alternate start sites at Met82 and Met286, while a third shows a four amino acid substitution for aa 1-34. Human NDRG1 shares 95% amino acid identity with mouse NDRG1 over amino acids 11-267 .

Where is NDRG1 typically expressed in human tissues?

NDRG1 demonstrates ubiquitous expression across multiple tissue types, particularly in fibroblasts, endothelial cells, respiratory epithelium, and prostate epithelium. It is predominantly expressed in nonproliferating, differentiating tissues under normal physiological conditions . In cancer contexts such as bladder cancer, NDRG1 is mainly expressed in the cytoplasm (62%) and on cell membranes (38%), with some nuclear localization also observed .

How should researchers approach NDRG1 detection in experimental settings?

For optimal NDRG1 detection, researchers should consider:

• Western blotting: Use specific antibodies such as Goat Anti-Human NDRG1 Antigen Affinity-purified Polyclonal Antibody (1 μg/mL) followed by HRP-conjugated secondary antibodies. NDRG1 typically appears as a 43 kDa band under reducing conditions .

• Immunohistochemistry: Employ anti-NDRG1 antibody (1:600) with appropriate HRP-conjugated secondary antibodies. Quantify expression using the German semi-quantitative scoring system by multiplying intensity score (0-3) with the percentage score of stained cells .

• ELISA: Direct ELISA assays show less than 1% cross-reactivity with recombinant human N-myc .

What experimental approaches effectively modulate NDRG1 expression?

For reliable manipulation of NDRG1 expression:

• Overexpression: Transfect cells with pcDNA3.1-NDRG1 plasmid (based on GenBank: NM_001135242.1) using Attractene Transfection Reagent.

• Knockdown: Apply NDRG1 SignalSilence siRNAs with HiPerFect Transfection Reagent. Mixing multiple siRNAs (e.g., siRNA I: #6245 and siRNA II: #6257) can enhance knockdown efficiency.

• Validation timeline: Harvest cells 48 hours post-transfection for optimal expression changes .

What functional assays best evaluate NDRG1's biological impact?

Researchers should employ multiple complementary assays:

• Proliferation: CCK-8 assay to measure cell viability changes.

• Migration: Wound healing assay with measurements at 24h and 48h time points.

• Invasion: Transwell assays with crystal violet staining (48h for 5637 cells; 24h for T24 cells).

• Protein interactions: Co-immunoprecipitation to detect associations with key partners like γ-tubulin .

• EMT progression: Western blot and RT-PCR for epithelial markers (Cytokeratin 7, Claudin-1) and mesenchymal markers (N-cad, β-catenin, Slug) .

How does NDRG1 contribute to tumorigenesis and cancer progression?

NDRG1 demonstrates complex roles in cancer development:

• Expression patterns: Significantly increased NDRG1 mRNA and protein levels appear in bladder cancer tissues compared to paired tumor-free tissues.

• Cellular effects: NDRG1 overexpression correlates with increased cell proliferation, migration, and invasion while decreasing apoptotic cell numbers in bladder cancer models.

• EMT promotion: Upregulated NDRG1 expression associates with downregulated epithelial markers and upregulated mesenchymal markers, promoting a more aggressive cancer phenotype .

What is the prognostic significance of NDRG1 expression in human cancers?

NDRG1 serves as a valuable prognostic indicator:

What is the relationship between NDRG1 and TP53 in cancer development?

A complex mutual exclusivity exists between these factors:

• Genomic analysis: Homozygous loss of TP53 is nearly mutually exclusive with NDRG1 overexpression in over 96% of human cancers.

• Functional connection: TP53-null cells fail to increase NDRG1 expression under physiologic low-proliferating conditions, unlike wild-type controls and TP53 R248W knockin cells.

• Mechanistic significance: This relationship provides insight into how TP53 loss leads to genomic instability through disruption of NDRG1-mediated centrosome homeostasis .

How does NDRG1 regulate genomic stability?

NDRG1 maintains genomic integrity through specific mechanisms:

• Centrosome regulation: NDRG1 physically associates with γ-tubulin, a key centrosome component, controlling centrosome number and preventing amplification.

• p53 dependency: Under physiologic low proliferative states, p53 induces NDRG1 expression, which then helps maintain centrosome homeostasis.

• Genomic consequences: Loss of TP53 leads to failure in upregulating NDRG1, resulting in supernumerary centrosomes and potential aneuploidy.

• Cancer relevance: This mechanism helps explain how cells with low proliferative index and p53 loss acquire additional genetic alterations leading to cancer .

What role does NDRG1 play in reproductive biology?

NDRG1 serves critical functions in ovarian physiology:

• Expression pattern: NDRG1 is absent in primary follicles but present in all granulosa cells beyond the tertiary stage.

• Regulatory relationship: HIF-1α regulates NDRG1 expression, with HIF-1α siRNA significantly decreasing NDRG1 at both mRNA and protein levels.

• Functional significance: HIF-1α and NDRG1 are integral to follicular development and early luteinization of pre-ovulatory follicles.

• Hormonal impact: NDRG1 reduction correlates with decreased progesterone synthesis, suggesting a role in steroidogenesis .

How might differential expression of NDRG1 isoforms impact experimental outcomes?

Researchers should consider isoform-specific effects:

• Known variants: Three potential isoform variants exist - two with alternate start sites (Met82 and Met286) and one with a four amino acid substitution for positions 1-34.

• Experimental considerations: When designing expression constructs or knockout strategies, researchers must account for these variants to ensure complete targeting.

• Antibody selection: Antibodies targeting different protein regions may detect specific isoforms preferentially. For comprehensive detection, researchers should select antibodies recognizing conserved regions .

What are the limitations of current NDRG1 detection methods?

Current methodological challenges include:

• Subcellular localization: NDRG1 demonstrates variable localization across cytoplasmic, membrane, and nuclear compartments, requiring compartment-specific isolation protocols.

• Cross-reactivity: Some antibodies show minor cross-reactivity with N-myc, necessitating careful validation.

• Tissue heterogeneity: Variable expression across cell types within tissues may complicate interpretation of bulk tissue analyses.

• Post-translational modifications: Current methods may not distinguish between modified and unmodified NDRG1 forms .

How should researchers approach contradictory findings regarding NDRG1's role?

When encountering conflicting results:

• Cell-type specificity: Examine whether contradictions arise from different cell types or tissues, as NDRG1 function varies contextually.

• Disease stage consideration: NDRG1's role may evolve during disease progression; early protective effects may differ from later stage impacts.

• p53 status: Always determine and report TP53 status, as this significantly influences NDRG1 function.

• Isoform expression: Verify which NDRG1 isoforms predominate in the experimental system.

• Proliferation state: Document proliferation rates, as NDRG1's function differs between high and low proliferative states .

What are the most promising therapeutic applications targeting NDRG1?

Based on current evidence:

• Cancer biomarkers: NDRG1 shows promise as a diagnostic and prognostic biomarker, particularly in bladder cancer.

• Centrosome stabilization: In TP53-null cancers, restoring NDRG1 function might reduce genomic instability.

• EMT inhibition: Targeting NDRG1 could potentially reduce cancer invasion and metastasis by inhibiting EMT.

• Personalized approaches: Therapeutic strategies should consider the nearly mutual exclusivity between TP53 loss and NDRG1 overexpression .

Which methodological advancements would most benefit NDRG1 research?

Future technical developments should prioritize:

• Isoform-specific tools: Development of isoform-selective antibodies and expression constructs.

• Live-cell imaging: Methods to track NDRG1 dynamics during cell cycle progression and centrosome duplication.

• High-throughput screening: Identification of small molecules that specifically modulate NDRG1 function.

• Tissue-specific conditional models: Development of systems to study tissue-specific NDRG1 function in vivo.

• Single-cell approaches: Technologies to examine NDRG1 expression and function at the single-cell level within heterogeneous tissues .