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An initial characterisation of the Unfolded Protein Response pathway in haematopoietic canine cancer cell lines – a necessary step for the future development of new therapies in dogs with neoplasia

Beatriz Hernández-Suárez
Beatriz Hernández-Suárez
, 
David A. Gillespie
David A. Gillespie
, 
Bożena Obmińska-Mrukowicz
Bożena Obmińska-Mrukowicz
 e 
Aleksandra Pawlak
Aleksandra Pawlak
   | 20 set 2023
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Pubblicato online: 20 set 2023
Pagine: 447 - 458
Ricevuto: 28 mar 2023
Accettato: 02 ago 2023
DOI: https://doi.org/10.2478/jvetres-2023-0042
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© 2023 Beatriz Hernández-Suárez et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

Unfolding protein response pathway scheme. ER – endoplasmic reticulum; PERK – protein kinase RNA-like ER kinase; eIF2α – eukaryotic translation initiation factor 2α; p-eIF2α – phosphorylated eukaryotic translation initiation factor 2α; GRP78 – glucose-regulated protein 78; ATF4 – activating transcription factor 4; CHOP – CCAAT/enhancer binding protein homologous protein
Unfolding protein response pathway scheme. ER – endoplasmic reticulum; PERK – protein kinase RNA-like ER kinase; eIF2α – eukaryotic translation initiation factor 2α; p-eIF2α – phosphorylated eukaryotic translation initiation factor 2α; GRP78 – glucose-regulated protein 78; ATF4 – activating transcription factor 4; CHOP – CCAAT/enhancer binding protein homologous protein

Fig. 2.

Relative gene contents of the unfolded protein response (UPR) pathway and other pathways among genes expressed in the CLBL-1 B-cell lymphoma and GL-1 B-cell leukaemia canine cell lines (A) and relative expression of principal genetic components of the UPR pathway normalised to ACTB gene expression (B). ATF4 – activated transcription factor 4; EIF2A – eukaryotic translation initiation factor 2α; HSPA5 – heat-shock 70 kDa protein 5; DDIT3 – DNA-damage–inducible transcript 3; EIF2AK3 – eukaryotic translation initiation factor 2α kinase 3
Relative gene contents of the unfolded protein response (UPR) pathway and other pathways among genes expressed in the CLBL-1 B-cell lymphoma and GL-1 B-cell leukaemia canine cell lines (A) and relative expression of principal genetic components of the UPR pathway normalised to ACTB gene expression (B). ATF4 – activated transcription factor 4; EIF2A – eukaryotic translation initiation factor 2α; HSPA5 – heat-shock 70 kDa protein 5; DDIT3 – DNA-damage–inducible transcript 3; EIF2AK3 – eukaryotic translation initiation factor 2α kinase 3

Fig. 3.

Expression of unfolded protein response (UPR) proteins by canine lymphoma (CLBL-1 – B-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin in order to induce endoplasmic reticulum stress (A). Expression levels were measured by Western blot and densitometry and the densitometric quantification of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) was normalised to β-actin. Fragment of human and canine gene and eIF2α protein sequences surrounding residue 51, showing the conserved serine (phosphorylation site when activation of the protein takes place) (B). CHOP – CCAAT/EBP enhancer binding protein homologous protein; GADD153 – growth-arrest and DNA-damage–inducible gene 153; cc – control; tg – thapsigargin; eto – etoposide; bp – base pairs; aa – amino acids
Expression of unfolded protein response (UPR) proteins by canine lymphoma (CLBL-1 – B-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin in order to induce endoplasmic reticulum stress (A). Expression levels were measured by Western blot and densitometry and the densitometric quantification of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) was normalised to β-actin. Fragment of human and canine gene and eIF2α protein sequences surrounding residue 51, showing the conserved serine (phosphorylation site when activation of the protein takes place) (B). CHOP – CCAAT/EBP enhancer binding protein homologous protein; GADD153 – growth-arrest and DNA-damage–inducible gene 153; cc – control; tg – thapsigargin; eto – etoposide; bp – base pairs; aa – amino acids

Fig. 4.

Expression of CCAAT enhancer-binding protein homologous protein (CHOP) by canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma), canine leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) and human osteosarcoma (U2OS) cell lines treated with thapsigargin and MG132 in order to induce endoplasmic reticulum stress; expression being detected by a murine (CHOP (growth-arrest and DNA-damage–inducible gene 153 (GADD153) clone B-3) and a human (CHOP clone L637F) antibody (A). Quantification was assessed by densitometry. (B) Murine–human species epitope differences between CHOP/GADD153 B-3 and CHOP L637F with indication of the greater homology at the protein level between dogs and humans than between dogs and mice. CHOP L637F antibody immunogen surrounds amino acid 158, circled in red. cc – control; tg – thapsigargin; eto – etoposide; aa – amino acid; Ala – alanine
Expression of CCAAT enhancer-binding protein homologous protein (CHOP) by canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma), canine leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) and human osteosarcoma (U2OS) cell lines treated with thapsigargin and MG132 in order to induce endoplasmic reticulum stress; expression being detected by a murine (CHOP (growth-arrest and DNA-damage–inducible gene 153 (GADD153) clone B-3) and a human (CHOP clone L637F) antibody (A). Quantification was assessed by densitometry. (B) Murine–human species epitope differences between CHOP/GADD153 B-3 and CHOP L637F with indication of the greater homology at the protein level between dogs and humans than between dogs and mice. CHOP L637F antibody immunogen surrounds amino acid 158, circled in red. cc – control; tg – thapsigargin; eto – etoposide; aa – amino acid; Ala – alanine

Fig. 5.

Apoptosis in canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin and MG132 to induce endoplasmic reticulum stress and stained with annexin V. The upper left quadrants represent necrotic cells, the bottom left quadrants the living cell population, the upper right quadrants late apoptotic events and the bottom right quadrants early apoptotic events (A). Apoptosis in the same cell lines after the same treatments indicated by caspase 3/7 activation, confirming no major increase in apoptosis (except for in the CLBL-1 cell line). The second peak corresponds to apoptotic cells which harbour an active form of caspase 3/7. The red values represent means ± standard deviation (B). cc – control; FITC – fluorescein isothiocyanate
Apoptosis in canine lymphoma (CLBL-1 – B-cell lymphoma and CNK-89 – natural-killer-cell lymphoma) and leukaemia (CLB70 – B-cell chronic lymphocytic leukaemia and GL-1 – B-cell leukaemia) cell lines treated with thapsigargin and MG132 to induce endoplasmic reticulum stress and stained with annexin V. The upper left quadrants represent necrotic cells, the bottom left quadrants the living cell population, the upper right quadrants late apoptotic events and the bottom right quadrants early apoptotic events (A). Apoptosis in the same cell lines after the same treatments indicated by caspase 3/7 activation, confirming no major increase in apoptosis (except for in the CLBL-1 cell line). The second peak corresponds to apoptotic cells which harbour an active form of caspase 3/7. The red values represent means ± standard deviation (B). cc – control; FITC – fluorescein isothiocyanate

Gene Ontology knowledge base sets selected for the analysis

Gene set Species
GOBP_REGULATION_OF_PERK_MEDIATED_UNFOLDED_PROTEIN_RESPONSE human
GOBP_REGULATION_OF_PERK_MEDIATED_UNFOLDED_PROTEIN_RESPONSE mouse
HALLMARK_UNFOLDED_PROTEIN_RESPONSE human
HALLMARK_UNFOLDED_PROTEIN_RESPONSE mouse
REACTOME_UNFOLDED_PROTEIN_RESPONSE_UPR human
REACTOME_UNFOLDED_PROTEIN_RESPONSE_UPR mouse
WP_PHOTODYNAMIC_THERAPYINDUCED_UNFOLDED_PROTEIN_RESPONSE human
WP_UNFOLDED_PROTEIN_RESPONSE human

Antibody list showing the percentage of protein identity between humans and dogs

Protein Clone Supplier and catalogue No. Dilution used in the study % homology*
Anti-gamma H2AX 9F3 Abcam, Cambridge, UK, ab26350 1 : 1,000 in 3% BSA in TBS-T 99.17
Phospho-elF2α (Ser51) 119A11 Cell Signaling Technologies, Danvers, MA, USA, 3597 1 : 1,000 in 3% BSA in TBS-T 96.07
eIF2α D-3 Santa Cruz Biotechnology, Dallas, TX, USA, sc-133132 1 : 1,000 in 3% BSA in TBS-T 96.07
DDIT3/CHOP/GADD153 B-3 Santa Cruz Biotechnology, sc-7351 1 : 500 in 3% BSA in TBS-T 92.3
CHOP L637F Cell Signaling Technologies, 2895 1 : 1,000 in 3% BSA in TBS-T 92.3
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