The effect of Zhangfei/CREBZF on cell growth, differentiation, apoptosis, migration, and the unfolded protein response in several canine osteosarcoma cell lines

Abstract

Background: We had previously shown that the bLZip domain-containing transcription factor, Zhangfei/CREBZF inhibits the growth and the unfolded protein response (UPR) in cells of the D-17 canine osteosarcoma (OS) line and that the effects of Zhangfei are mediated by it stabilizing the tumour suppressor protein p53. To determine if our observations with D-17 cells applied more universally to canine OS, we examined three other independently isolated canine OS cell lines--Abrams, McKinley and Gracie.

Results: Like D-17, the three cell lines expressed p53 proteins that were capable of activating promoters with p53 response elements on their own, and synergistically with Zhangfei. Furthermore, as with D-17 cells, Zhangfei suppressed the growth and UPR-related transcripts in the OS cell lines. Zhangfei also induced the activation of osteocalcin expression, a marker of osteoblast differentiation and triggered programmed cell death.

Conclusions: Osteosarcomas are common malignancies in large breeds of dogs. Although there has been dramatic progress in their treatment, these therapies often fail, leading to recurrence of the tumour and metastatic spread. Our results indicate that induction of the expression of Zhangfei in OS, where p53 is functional, may be an effective modality for the treatment of OS.

Figure 1

p53 in dog osteosarcoma cell lines. (A) Schematic structure of full-length p53. TAD: N-terminal transactivation domain; PRR: proline-rich region; p53C: central DNA-binding domain; TET: tetramerization domain; CT: extreme carboxyl terminus. p53C is the domain where most cancer-associated p53 mutations are located. The numbers below the diagram indicate amino acid residues delineating the domains and numbers above the diagram represent the residues with highest frequency of oncogenic missense mutations [20]. (B) Derived amino acid sequence alignment of p53s from 4 dog OS cell lines and dog wild-type p53. The residues that have high mutant frequency were marked above the diagram. Accession numbers: KP279761, KP279762, KP279763, KP279764 (C) p53 proteins of dog OS cell lines have transcriptional activity, and Zhangfei enhances p53-dependent transactivation. D–17, Abrams, McKinley, and Gracie cells were transfected with 0.5 μg of pCAT3B or pCAT3B-p53RE, in the presence or absence of 1 μg of pcZF. 24 h after transfection, the CAT activity was determined. Values represented the relative CAT activity (adjusted by β-galactosidase) of different treatments. Standard deviations from means of three individual experiments are shown. Significance of differences of the means (*P < 0.05, **P < 0.01) were determined using ANOVA.

Figure 2

Ectopic expression of Zhangfei suppresses cell growth in canine osteosarcomas. D–17, Abrams, McKinley, and Gracie canine OS cells were mock-infected or infected with adenovirus vectors expressing either Zhangfei (Adeno-ZF) or β-galactosidase (Adeno-LacZ) and growth rates were measured (A) by absorbance at 405 nm with WST-1 at different time points after infection. Error bars indicate standard deviations from means of three individual experiments. Standard deviations from means of three individual experiments are shown and significance (*P < 0.05, **P < 0.01) was determined using ANOVA. (B). Zhangfei was detected by immunoblotting using antiserum against Zhangfei.

Figure 3

Zhangfei causes canine osteosarcoma cells to commit apoptosis. D–17, Abrams, McKinley and Gracie cells, mock-infected or infected with Adeno-ZF or Adeno-LacZ or treated with 50 μM etoposide (positive control) were stained with fluorescent Annexin V and propidium iodide. Unstained cells or cells staining with either or both dyes were enumerated by FACS. A4 represents the percentage of total cells undergoing apoptosis. For McKinley and Gracie only reading for 48 hr are shown. Differences between LacZ and ZF-expressing cells were more pronounced at that time-point.

Figure 4

Zhangfei induces differentiation of canine osteosarcoma cells. D–17 and Abrams cells were either mock-infected or infected with Adeno-ZF or Adeno-LacZ. The positive control cells were treated with 10^-5 mM vitamin D3. The mRNA levels of osteoblast differentiation marker (osteocalcin) were estimated by qRT-PCR. Standard deviations from means and P values as calculated using a Student T-test are shown.

Figure 5

Ectopic expression of Zhangfei causes decreased ability of canine osteosarcoma cells to repair a scratch wound. (A) Scratch wounds were made in 100% confluent cultures of D–17 or Abrams cells mock-infected or infected with Adeno-ZF or Adeno-LacZ. Phase contrast images were taken at 0, 4, 8, 12, and 24 hours after infection from identical regions. (B) The wound size relative to the starting wound size was measured at each time point after infection in three independent experiments and expressed as a percentage reduction in wound size + standard deviation. Significance of differences of means (*P < 0.05, **P < 0.01) were determined using ANOVA.

Figure 6

Zhangfei negatively regulates the Unfolded Protein Responses (UPR) in canine osteosarcomas. (A) D–17 cells were left untreated, treated with thapsigargin or grown in glucose free medium. Twelve hours later cells were harvested and transcripts for the UPR- linked genes Xbp1s, HERP, CHOP and GRP78 estimated by qRT-PCR. Cells were mock-infected or infected with either Adeno-ZF or Adeno-LacZ and then treated with thapsigargin (B) or deprived of glucose (C). 24 hr later cells were harvested and transcripts for UPR genes estimated. Proteins in thapsigargin-treated mock-infected and Adeno-ZF-infected cells were detected by immunoblots (D) and immunofluorescence (E). Dotted lines in A and B indicate a 2-fold difference. Values greater than 2 fold were considered significant.