BIRC7 Antibody

What is BIRC7 and what is its biological significance?

BIRC7 (also known as Livin, KIAP, ML-IAP, MLIAP, or RNF50) is a member of the inhibitor of apoptosis proteins (IAP) family. It contains a single baculoviral IAP repeat (BIR) domain that is homologous to the BIR3 domain of other IAPs, and a RING (Really Interesting New Gene) zinc finger domain .

The protein plays a critical role in inhibiting apoptosis by binding to and inhibiting cell death proteases, particularly caspases-3, -7, and -9 . This inhibition of caspase activation is a key mechanism by which BIRC7 prevents programmed cell death, making it significant in cancer research where dysregulation of apoptosis is common .

BIRC7 exists in two isoforms: Livin-alpha and Livin-beta, which are almost identical except for 18 amino acids located in the BIR-RING interlinking region present only in the alpha isoform . Despite this high similarity, the two isoforms are involved in anti-apoptotic responses to different stimuli.

How is BIRC7 expression distributed in normal and pathological tissues?

BIRC7 shows a distinctive expression pattern across tissues:

Normal tissues:

• Low expression in fetal kidney, fetal liver, testis, and thymus

• Generally not detectable in normal thyroid epithelial cells (Nthy-ori-3-1 cell line)

• Limited expression in normal adrenal tissues

Pathological tissues:

• Highly expressed in melanomas

• Significantly upregulated in papillary thyroid carcinoma (PTC) compared to matched normal tissues

• Elevated expression in adrenocortical carcinomas compared to adenomas and normal adrenal glands

• Expression correlates with increased lymph node metastasis in PTC

The differential expression between normal and cancerous tissues makes BIRC7 a potential biomarker for several malignancies and suggests its role in tumor development.

What methodologies are available for BIRC7 detection, and which applications are most suitable for different research questions?

BIRC7 can be detected using several techniques with specific antibodies:

When selecting an antibody and method, researchers should consider:

• The specific isoform of interest (alpha or beta)

• The cellular localization being studied

• The need for quantitative versus qualitative data

• The sample type (fresh-frozen vs. paraffin-embedded)

How can researchers validate the specificity of BIRC7 antibodies?

To ensure antibody specificity for BIRC7:

• Positive and negative controls: Use cell lines with known BIRC7 expression levels. For positive controls, melanoma cell lines or transfected cells overexpressing BIRC7 are recommended. Normal thyroid epithelial cells (Nthy-ori-3-1) can serve as negative controls .

• Western blot validation: Verify the molecular weight (approximately 30-32 kDa, although some antibodies may detect bands at ~35 kDa or even 68 kDa) . Note that a secondary band at approximately 30 kDa may represent the short form of Livin .

• Knockdown/overexpression verification: Perform antibody testing in BIRC7 knockdown (siRNA or shRNA) and overexpression models to confirm specificity .

• Cross-reactivity assessment: Check for potential cross-reactivity with other IAP family members. Many commercial antibodies specify "no cross-reactivity with other proteins" .

• Epitope mapping: Consider the specific region of BIRC7 targeted by the antibody. Different antibodies target different regions (N-terminal, C-terminal, or specific amino acid sequences) .

How does BIRC7 contribute to cancer progression and metastasis?

BIRC7 plays multifaceted roles in cancer progression:

• Apoptosis inhibition: By inhibiting caspases-3, -7, and -9, BIRC7 prevents programmed cell death, allowing cancer cells to evade apoptosis .

• Promotion of epithelial-mesenchymal transition (EMT): Studies in papillary thyroid carcinoma show that BIRC7 enhances cell invasion and migration by promoting EMT. Knockdown of BIRC7 significantly decreased PTC cell migration and invasion, while overexpression enhanced these capabilities .

• Autophagy suppression: BIRC7 suppresses autophagy in PTC cells by modulating the expression of ATG5 and BECN1. This suppression contributes to enhanced EMT and metastasis .

• Response to hypoxia: BIRC7 is upregulated under hypoxic conditions and acts as a downstream factor of HIF-1α in melanoma cells. Knockdown of BIRC7 blocks the promoting effect of hypoxia on cell proliferation .

• Malignancy marker: In adrenocortical tumors, BIRC7 expression correlates with malignancy, suggesting its potential as a diagnostic marker .

What is the relationship between BIRC7 and hypoxia in tumor microenvironments?

BIRC7 plays a crucial role in the hypoxic response of cancer cells:

• Transcriptional regulation by HIF-1α: Hypoxia significantly increases BIRC7 expression at both mRNA and protein levels. HIF-1α acts as a transcription factor for BIRC7, binding to its promoter and enhancing transcription .

• Functional significance: Experimental data from melanoma cells (A875 and M14) show that:

  • Hypoxia promotes cell proliferation and inhibits apoptosis

  • These effects are significantly reversed by BIRC7 knockdown

  • BIRC7 is essential for the pro-tumorigenic effects of hypoxia

• Potential therapeutic implications: Targeting BIRC7 in hypoxic tumor microenvironments may counteract the effects of hypoxia on tumor progression. This approach could be particularly relevant for melanomas, which often contain hypoxic regions .

The experimental evidence suggests that BIRC7 is not merely upregulated in hypoxia but is functionally important for mediating hypoxia-induced cancer progression.

How can BIRC7 be effectively targeted in experimental models?

Several approaches for manipulating BIRC7 in research models include:

• RNA interference:

  • siRNA transfection has been successfully used to downregulate BIRC7 expression in multiple cancer cell lines, including melanoma (A875, M14) and papillary thyroid carcinoma cells (BCPAP, K1)

  • shRNA can provide more stable knockdown for long-term experiments

• Overexpression systems:

  • Transfection with BIRC7-overexpressing constructs enables gain-of-function studies

  • Both isoforms (alpha and beta) can be separately overexpressed to study their distinct functions

• HIF-1α inhibitors:

  • In hypoxia studies, HIF-1α inhibitors can be used to modulate BIRC7 expression indirectly

• Combined approaches:

  • Targeting both BIRC7 and mTOR enhances autophagy in PTC cells and achieves synergistic antimetastatic efficacy both in vitro and in vivo

• In vivo models:

  • Tumor xenograft models have been used to study BIRC7 function in cancer progression and metastasis

What techniques are most effective for studying BIRC7 isoform-specific functions?

The two BIRC7 isoforms (alpha and beta) have distinct functions despite their high sequence similarity. To study isoform-specific functions:

• Isoform-specific primers for qRT-PCR:

  • Design primers that can distinguish between alpha and beta isoforms based on the 18 amino acid difference

  • This approach has been used to evaluate isoform expression in adrenocortical tissues

• Isoform-specific antibodies:

  • Some commercial antibodies may recognize epitopes specific to one isoform

  • Western blotting can sometimes distinguish isoforms based on slight molecular weight differences

• Expression vectors:

  • Create expression vectors containing only one isoform (alpha or beta)

  • Transfect cells to study the specific effects of each isoform on cellular processes

• Functional assays to assess differential responses:

  • Expose cells expressing specific isoforms to different apoptotic stimuli

  • Livin-alpha is associated with resistance against staurosporine

  • Livin-beta is associated with resistance to etoposide, UV irradiation, and TNF-α induced apoptosis

• Tissue distribution analysis:

  • Compare the distribution of isoforms across different tissues to identify context-specific functions

How can researchers effectively analyze BIRC7's role in autophagy regulation?

To investigate BIRC7's role in autophagy:

• Autophagy marker analysis: Western blotting for key autophagy markers:

  • LC3-I to LC3-II conversion (increased LC3II indicates enhanced autophagy)

  • P62/SQSTM1 levels (decreased levels indicate enhanced autophagy)

  • BIRC7 knockdown has been shown to increase LC3II and reduce P62 levels, while BIRC7 overexpression shows opposite effects

• Fluorescent reporter systems:

  • Transfection with adenoviral mRFP-GFP-LC3 reporter vector

  • In BIRC7 knockdown cells, increased RFP-positive red puncta indicate enhanced autophagic flux

  • BIRC7 overexpression reduces these autophagy indicators

• Transmission electron microscopy (TEM):

  • Direct visualization of autophagosomes

  • BIRC7 knockdown significantly induces autophagosome formation

  • BIRC7 overexpression decreases autophagosome formation

• Autophagy modulator experiments:

  • Combine BIRC7 manipulation with autophagy inducers (rapamycin, starvation) or inhibitors (chloroquine, 3-methyladenine)

  • Assess whether these modulators can reverse or enhance BIRC7's effects on autophagy

• Mechanistic studies:

  • Investigate BIRC7's effects on autophagy regulators such as ATG5 and BECN1

  • Explore the relationship between BIRC7, mTOR, and autophagy in cancer progression

What are the best experimental designs for investigating BIRC7's role in apoptosis inhibition?

To effectively study BIRC7's anti-apoptotic functions:

• Apoptosis assays following BIRC7 manipulation:

  • Flow cytometry with Annexin V/PI staining to quantify apoptotic cell populations

  • BIRC7 knockdown has been shown to increase the percentage of apoptosis in cancer cells under both normoxia and hypoxia

  • CCK-8 assays to assess cell viability and proliferation after BIRC7 manipulation

• Caspase activity assays:

  • Measure the activity of caspases-3, -7, and -9 (known BIRC7 targets)

  • Compare activities in cells with normal, overexpressed, or knocked-down BIRC7 levels

  • Use fluorogenic substrates specific for each caspase

• Protein interaction studies:

  • Co-immunoprecipitation to confirm direct interactions between BIRC7 and specific caspases

  • Investigate whether the BIR domain is responsible for these interactions

• Apoptosis induction models:

  • Test BIRC7's protective effects against various apoptotic stimuli:

    • Death receptor ligands (TNF-α, TRAIL)

    • Chemotherapeutic agents

    • UV irradiation

    • Serum starvation

  • These models can also distinguish between isoform-specific effects (alpha vs. beta)

• In vivo tumor models:

  • Xenograft models with BIRC7-manipulated cancer cells

  • Assess tumor growth rates, apoptotic indices, and responses to therapy

How is BIRC7 being investigated as a potential therapeutic target in cancer?

BIRC7 presents several promising avenues for therapeutic targeting:

• Direct BIRC7 inhibition strategies:

  • Small molecule inhibitors targeting the BIR domain to prevent caspase binding

  • Peptide-based mimetics that compete with BIRC7 for caspase binding

  • SMAC mimetics that can antagonize multiple IAP family members including BIRC7

• Transcriptional regulation:

  • HIF-1α inhibitors may indirectly reduce BIRC7 expression in hypoxic tumors

  • Investigation of other transcriptional regulators of BIRC7 expression

• Combination therapeutic approaches:

  • Targeting both BIRC7 and mTOR enhances autophagy in cancer cells and achieves synergistic antimetastatic efficacy

  • Combining BIRC7 inhibition with conventional chemotherapy or radiotherapy to overcome resistance

• Biomarker potential:

  • BIRC7 expression as a prognostic marker in multiple cancer types

  • Development of diagnostic tools based on BIRC7 expression patterns

• Isoform-specific targeting:

  • Developing therapeutic approaches that specifically target either the alpha or beta isoform based on context-specific functions

  • This could potentially reduce side effects by preserving normal BIRC7 functions

What are the current challenges in BIRC7 antibody-based research and potential solutions?

Researchers face several challenges when working with BIRC7 antibodies:

• Distinguishing between isoforms:

  • Challenge: The high sequence similarity between alpha and beta isoforms makes specific detection difficult

  • Solution: Development of isoform-specific antibodies targeting the 18 amino acid region unique to the alpha isoform

• Molecular weight variations in detection:

  • Challenge: Different studies report varying molecular weights (30-68 kDa) for BIRC7

  • Solution: Careful validation with positive controls and isoform-specific constructs; awareness that post-translational modifications may affect apparent molecular weight

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other IAP family members

  • Solution: Thorough validation using knockout/knockdown models and multiple detection methods

• Tissue-specific expression patterns:

  • Challenge: Variable expression across tissues affects detection sensitivity

  • Solution: Optimize protocols for specific tissue types; use appropriate positive controls

• Reproducibility issues:

  • Challenge: Variability in results across different antibody sources

  • Solution: Standardized validation protocols and reporting of antibody validation data; use of multiple antibodies targeting different epitopes for confirmation

What are the optimal storage and handling conditions for BIRC7 antibodies?

To maintain antibody integrity and performance:

• Storage temperature:

  • Store antibodies at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Working solution preparation:

  • For short-term use (up to three months), antibodies can be stored at 4°C

  • Most commercial BIRC7 antibodies are supplied in PBS containing preservatives like 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting practice:

  • Divide antibody stock into small aliquots to minimize freeze-thaw cycles

  • Store working dilutions separately from stock solutions

• Temperature considerations during procedures:

  • Keep antibodies on ice during experimental procedures

  • Avoid exposure to high temperatures which can cause denaturation

• Safety considerations:

  • Be aware that many antibody solutions contain sodium azide, which is toxic

  • Follow appropriate safety protocols for handling and disposal

How can researchers optimize immunohistochemistry protocols for BIRC7 detection in different tissue types?

For optimal IHC results with BIRC7 antibodies:

• Antigen retrieval optimization:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • For BIRC7, heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly effective

• Antibody dilution titration:

  • Most commercial BIRC7 antibodies work in the range of 1:50-1:200 for IHC applications

  • Perform dilution series to determine optimal concentration for specific tissue types

• Incubation conditions:

  • Test different incubation times and temperatures (overnight at 4°C vs. 1-2 hours at room temperature)

  • Use humidity chambers to prevent tissue drying

• Detection system selection:

  • For samples with low BIRC7 expression, amplification systems may improve sensitivity

  • Compare DAB vs. fluorescent detection based on research needs

• Tissue-specific considerations:

  • Melanoma tissues typically show higher BIRC7 expression and may require higher antibody dilutions

  • Normal tissues with low BIRC7 expression may require more sensitive detection methods

  • For adrenocortical tumors, background staining should be carefully controlled

By addressing these technical considerations, researchers can obtain more reliable and reproducible results when studying BIRC7 across various experimental contexts.