DIRAS1 Antibody

What is DIRAS1 and how does it differ from other Ras-family GTPases?

The gene encoding DIRAS1 is located on chromosome 19p13.3 and consists of 2 exons . DIRAS1 is closely related to its family members DiRas2 (predominantly expressed in brain) and DiRas3 (ARHI, a tumor suppressor in breast and ovarian cancers) .

What is the mechanism by which DIRAS1 functions as a tumor suppressor?

DIRAS1 exerts its tumor-suppressive effects through a unique mechanism that involves:

• Binding to SmgGDS, a protein that promotes the activation of oncogenic GTPases

• Inhibiting SmgGDS binding to other small GTPases, including K-Ras4B, RhoA, and Rap1A

• Acting as a dominant-negative small GTPase by sequestering SmgGDS

Even when DIRAS1 cDNA was transfected at only 25% of the molar ratio of the cDNA encoding pro-oncogenic small GTPases, it potently inhibited the detectable interactions of SmgGDS with RhoA, K-Ras4B, and Rap1A .

Additional tumor suppressive mechanisms include:

• Inhibition of RhoA- and SmgGDS-mediated NF-κB transcriptional activity

• Suppression of basal NF-κB activation in cancer cell lines

• Reduction of matrix metalloproteinase 2/9 expression

• Diminishment of BAD serine phosphorylation, promoting cell death

• Decrease in ERK1/2 and MAPK-mediated signals

What is the expression pattern of DIRAS1 in normal tissues?

DIRAS1 is expressed at high levels in brain and heart tissues . In normal brain tissue, DIRAS1 is expressed in glial cells, neurons, and microvasculature of the cerebral cortex . In normal breast tissue, DIRAS1 shows robust expression with differential patterns:

• 13 of 15 normal breast apical ductal epithelium samples stained positively for DIRAS1

• Myoepithelial cells expressed particularly high levels, with a mean Immunoreactive Score (IRS) of 9.04

How is DIRAS1 expression altered in cancer tissues?

DIRAS1 expression is typically downregulated in multiple cancer types compared to corresponding normal tissues:

This consistent downregulation across multiple cancer types strongly supports DIRAS1's role as a tumor suppressor .

What epigenetic mechanisms regulate DIRAS1 expression in cancer?

DNA methylation plays a significant role in silencing DIRAS1 expression in cancer cells. Research in renal cell carcinoma has demonstrated:

• CpG islands in the DIRAS1 promoter region are aberrantly hypermethylated in RCC cell lines (ACHN, 786-O, and Caki-1)

• Treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, significantly increases DIRAS1 expression

• Bisulfite sequencing PCR (BSP) confirms the hypermethylation patterns

This epigenetic silencing through promoter methylation represents a common mechanism for DIRAS1 downregulation across multiple cancer types, suggesting potential therapeutic approaches targeting DNA methylation to restore DIRAS1 expression.

What are the primary applications for DIRAS1 antibodies in cancer research?

DIRAS1 antibodies are employed in multiple research applications:

• Immunohistochemistry (IHC):

  • Detection of DIRAS1 expression in tissue microarrays

  • Analysis of subcellular localization (membrane, cytoplasm, nucleus)

  • Recommended dilutions range from 1:50-1:200

• Western Blotting (WB):

  • Quantification of DIRAS1 protein expression (predicted band size: 22 kDa)

  • Verification of experimental DIRAS1 modulation

  • Typical dilutions around 1:500

• Immunofluorescence/Immunocytochemistry:

  • Subcellular localization studies

  • Co-localization with potential interacting partners

• Co-Immunoprecipitation:

  • Investigation of DIRAS1 interactions with proteins like SmgGDS

What technical considerations are important for DIRAS1 antibody specificity?

To ensure specific DIRAS1 detection, researchers should consider:

• Antibody validation:

  • Peptide competition assays: Co-incubation with the immunizing peptide should abolish specific staining

  • Positive controls: Normal brain tissue, breast tissue, HK-2 cells

  • Negative controls: Cancer cell lines with low DIRAS1 expression (U87, U251, MCF-7, T47D)

• IHC optimization:

  • Antigen retrieval: Heat-mediated retrieval in citrate buffer (pH 6.0)

  • Blocking: 10% normal goat serum for 30 minutes at room temperature

  • Detection: Biotinylated secondary antibody with HRP-conjugated systems

• Western blot parameters:

  • Protein loading: 5-30 μg of total protein

  • Molecular weight verification: DIRAS1 appears at 22 kDa

  • Controls: HeLa and HepG2 extracts have been successfully used

What methods can be used to experimentally restore DIRAS1 expression in cancer cells?

Researchers have successfully used several approaches to restore DIRAS1 expression in cancer models:

• RNA activation (RNAa):

  • Small activating dsRNAs targeting the DIRAS1 promoter

  • dsDIRAS1-755 has shown approximately 3.0-fold induction of DIRAS1 expression

  • Target sites range from -824 to -473 relative to the transcription start site

• Vector-based overexpression:

  • Construction of DIRAS1 expression vectors (e.g., pDIRAS1)

  • Insertion of DIRAS1 cDNA into expression vectors like pIRES2-EGFP

  • Transfection using Lipofectamine 2000 or similar reagents

• Epigenetic modifiers:

  • Treatment with 5-Aza-CdR (DNA methyltransferase inhibitor) at 10 μM for 96 hours

  • Reverses promoter hypermethylation to restore DIRAS1 expression

What functional changes occur when DIRAS1 expression is restored in cancer cells?

Restoration of DIRAS1 expression in cancer cells produces multiple anti-tumor effects:

• Reduced proliferation and tumor growth:

  • Decreased cell viability in CCK-8 assays

  • Reduced colony formation in clonogenicity assays

  • Smaller tumor volume and weight in xenograft models

• Increased apoptosis:

  • Promoted programmed cell death in cancer cell lines

• Inhibited migration and invasion:

  • 50% reduction in migratory and invading cells in transwell assays

  • Reversal of these effects when DIRAS1 is knocked down

• Altered signaling pathways:

  • Suppressed NF-κB transcriptional activity

  • Decreased IL-8 transcript levels

  • Inhibition of RhoA-mediated signaling

How can DIRAS1 protein-protein interactions be investigated?

Several methods are effective for studying DIRAS1 interactions with other proteins:

• Co-immunoprecipitation:

  • Demonstrates DIRAS1 binding to SmgGDS

  • Shows competition with other small GTPases

  • Requires non-denaturing conditions to preserve protein interactions

• In silico docking studies:

  • Predict binding interfaces between DIRAS1 and partners

  • Have shown DIRAS1 binding to SmgGDS similar to other small GTPases

  • Generate hypotheses about competition mechanisms

• Guanine nucleotide exchange assays:

  • Confirm SmgGDS does not mediate GDP/GTP exchange on DIRAS1

  • Verify DIRAS1 binds SmgGDS nonproductively

• Competitive binding assays:

  • Demonstrate DIRAS1 competition with other GTPases for SmgGDS binding

  • Show potent inhibition even at low DIRAS1 expression levels

How does subcellular localization affect DIRAS1 function?

DIRAS1 shows interesting localization patterns that may influence its function:

• In normal tissues, DIRAS1 localizes to:

  • Cell membrane (lipid-anchor, cytoplasmic side)

  • Nucleus (particularly in para-carcinoma cervical tissues)

• In cancer tissues:

  • Nuclear accumulation is often lost

  • This differential localization may impact DIRAS1's tumor suppressive functions

Research studying these localization patterns typically employs immunohistochemistry with DIRAS1 antibodies and carefully optimized protocols for subcellular visualization .