Immunohistochemical Expression Study of Proapoptotic BH3-Only Protein Bad in Canine Nonneoplastic Tissues and Canine Lymphomas

Dettwiler, Martina; Croci, M.; Vaughan, Lloyd; Guscetti, Franco

doi:10.1177/0300985813478212

Cited by 6 publications

(8 citation statements)

References 18 publications

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“…Bcl-2 family proteins have been assigned a central importance in the pathogenesis of various human cancer syndromes (Kelly and Strasser, 2011;Nys and Agostinis, 2012). Likewise, they have been implicated in the pathogenesis of diverse canine tumors including for instance hemangiosarcoma, mammary tumors, tumors of neural and hematopoetic origin (Sano et al, 2003;Kumaraguruparan et al, 2006a;Kumaraguruparan et al, 2006b;Yan et al, 2006;Murakami et al, 2008;Ide et al, 2010;Strefezzi et al, 2012;Dettwiler et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…To verify expression of the respective protein after induction, representative culture samples collected at 24h or 48h after start of induction were analysed by western blot as previously described with minor modifications (Dettwiler et al, 2013). Briefly, protein extracts were prepared by mechanical lysis of the yeast cells (mixed to equal volumes of a lysis buffer containing 100 mM Tris-HCl at pH 6.8, 10% (v/v) glycerol, 3% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and 1% (w/v) bromophenol blue using glass beads and a Vortex mixer at maximum speed for 2 min.…”

Section: Western Blotsmentioning

confidence: 99%

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Sequence and partial functional analysis of canine Bcl-2 family proteins

Brot

Schade

Croci

et al. 2016

Research in Veterinary Science

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Dogs present with spontaneous neoplasms biologically similar to human cancers. Apoptotic pathways are deregulated during cancer genesis and progression and are important for therapy.We have assessed the degree of conservation of a set of canine Bcl-2 family members with the human and murine orthologs. To this end, seven complete canine open reading frames were cloned in this family, four of which are novel for the dog, their sequences were analyzed, and their functional interactions were studied in yeasts. We found a high degree of overall and domain sequence homology between canine and human proteins. It was slightly higher than between murine and human proteins. Functional interactions between canine pro-apoptotic Bax and Bak and anti-apoptotic Bcl-xL, Bcl-w, and Mcl-1 were recapitulated in yeasts. Our data provide support for the notion that systems based on canine-derived proteins might faithfully reproduce Bcl-2 family member interactions known from other species and establish the yeast as a useful tool for functional studies.

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Section: Discussionmentioning

confidence: 99%

Section: Western Blotsmentioning

confidence: 99%

Sequence and partial functional analysis of canine Bcl-2 family proteins

Brot

Schade

Croci

et al. 2016

Research in Veterinary Science

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“…Since the use of antibodies raised against heterologous antigens, which is a recurrent situation in studies including companion animals, can be problematic (Keller et al, 2007;Dettwiler et al, 2013) and BAX-Ab2D2 does not cross-react with murine BAX (Hsu and Youle, 1997), all antibodies used here were thoroughly validated. They were formally demonstrated to react with the recombinant canine homologues and to label endogenous levels of protein in a semi-quantitative manner in UV-irradiated keratinocytes.…”

Section: Discussionmentioning

confidence: 99%

“…Canine normal keratinocytes (Bskin cell line) were grown in Dulbecco's Modified Eagles medium, irradiated with 100 mJ UVC light using a cross linker, harvested and processed for western blot analysis and immunohistochemistry as described by Dettwiler et al (2013).…”

Section: Cultured Keratinocytesmentioning

confidence: 99%

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Immunohistochemical expression of Bax and Bak in canine non-neoplastic tissues

Croci

Dettwiler

Vaughan

et al. 2013

The Veterinary Journal

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Apoptosis is critical for embryonic development, maintenance of tissue homeostasis and protection against malignant transformation. The Bcl-2 family of proteins plays a key role in intrinsic apoptosis by controlling the integrity of the outer mitochondrial membrane, and the multidomain pro-apoptotic Bcl-2 family members Bax and Bak are essential components of this pathway. The aim of this study was to provide data on the expression of these proteins in normal canine tissues. Two antibodies against Bax recognising different conformations of the protein and one antibody against Bak were validated by immunohistochemistry and immunoblotting using canine recombinant proteins and keratinocytes treated with ultraviolet light. The antibodies were used immunohistochemically to label a wide panel of histologically normal tissues assembled on tissue microarrays. In addition, a subset of the tissues was evaluated by Western blot analysis. Immunohistochemical and Western blot analyses revealed that both Bax and Bak are widely expressed in non-neoplastic tissues from adult dogs. Immunohistochemistry showed almost exclusively cytoplasmic labelling and prominent labelling of epithelial cells. In lymph nodes, immunohistochemical labelling was diffuse for both proteins and showed enhanced intensities in the mantle zones for Bax and the germinal centres for Bak. Strong reactivity for the active conformation of Bax was detected only in enterocytes and Leydig cells and in scattered lymphocytes. These data indicate widespread expression of Bax and Bak in normal canine tissues. Knowledge of the expression of Bax and Bak in normal tissues is a prerequisite in assessing the role of these proteins in canine neoplastic disease. AbstractApoptosis is critical for embryonic development, maintenance of tissue homeostasis and protection against malignant transformation. The Bcl-2 family of proteins plays a key role in intrinsic apoptosis by controlling the integrity of the outer mitochondrial membrane, and the multidomain pro-apoptotic Bcl-2 family members Bax and Bak are essential components of this pathway. The aim of this study was to provide data on the expression of these proteins in normal canine tissues. Two antibodies against Bax recognising different conformations of the protein and one antibody against Bak were validated by immunohistochemistry and immunoblotting using canine recombinant proteins and keratinocytes treated with ultraviolet light. The antibodies were used immunohistochemically to label a wide panel of histologically normal tissues assembled on tissue microarrays. In addition, a subset of the tissues was evaluated by Western blot analysis. Immunohistochemical and Western blot analyses revealed that both Bax and Bak are widely expressed in non-neoplastic tissues from adult dogs. Immunohistochemistry showed almost exclusively cytoplasmic labelling and prominent labelling of epithelial cells. In lymph nodes, immunohistochemical labelling was diffuse for both proteins and showed enhanced intensities in the mantl...

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Expression of Apoptosis‐regulating Proteins Bcl‐2 and Bax in Lymph Node Aspirates from Dogs with Lymphoma

Meichner

Fogle

English

et al. 2016

Veterinary Internal Medicne

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BackgroundDysregulated apoptosis is a hallmark of tumorigenesis, and is also involved in resistance to cytotoxic treatment, and might be relevant in lymphoma in dogs.Hypothesis/ObjectivesThat Bcl‐2/Bax expression patterns differ between lymphoma immunophenotypes, and that Bcl‐2/Bax ratio is correlated with prognosis.AnimalsFifty‐five client‐owned dogs with multicentric lymphoma and 5 healthy dogs.MethodsProspective, case–control study. We compared 3 methods (flow cytometry, qRT‐PCR, Western blot) for Bcl‐2 and Bax quantification in a subset of dogs. The effect of time on Bcl‐2/Bax ratios measured by flow cytometry was assessed in lymphoma cell lines. Immunophenotype and Bcl‐2/Bax expression by flow cytometry were determined in LN aspirates from all dogs with multicentric lymphoma compared to healthy dogs. Progression‐free survival (PFS) was retrospectively evaluated in a group of dogs all receiving similar treatment.ResultsBcl‐2/Bax ratios remain consistent for at least 5 days after sample collection. Bcl‐2/Bax ratio was higher in dogs with T‐cell lymphoma (TCL; median 0.97, range 0.37–1.36) compared to B‐cell lymphoma (BCL; median 0.36, range 0.07–1.45) (P < .0001) and normal dogs (median 0.36, range 0.21–0.48) (P = .0006), respectively. Dogs with Bcl‐2/Bax ratios higher than the median of the group experienced a median PFS of 101 days and dogs with ratios equal and lower than the median had PFS of 130 days (P = .19).Conclusions and clinical importanceHigher intrinsic resistance to apoptosis following cytotoxic treatment might contribute to the less favorable prognosis associated with multicentric TCL in dogs. Whether Bcl‐2/Bax will be helpful to identify canine BCL and TCL with more aggressive and more indolent behavior, respectively, should be evaluated in larger prospective clinical studies.

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Immunohistochemical Expression Study of Proapoptotic BH3-Only Protein Bad in Canine Nonneoplastic Tissues and Canine Lymphomas

Cited by 6 publications

References 18 publications

Sequence and partial functional analysis of canine Bcl-2 family proteins

Sequence and partial functional analysis of canine Bcl-2 family proteins

Immunohistochemical expression of Bax and Bak in canine non-neoplastic tissues

Expression of Apoptosis‐regulating Proteins Bcl‐2 and Bax in Lymph Node Aspirates from Dogs with Lymphoma

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