Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a proapoptotic cytokine implicated in cancer cell surveillance. TRAIL induces apoptosis of target cells by the extrinsic, receptor-mediated, apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy agents. Development of TRAIL-resistant clones in the originally TRAIL-sensitive tumor cell population may be a critical complication of TRAIL based cancer therapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, “wild-type” (WT) HL60 cells, to recombinant soluble TRAIL resulted in the establishment of resistant subclones. To get deeper insight into the molecular mechanism of acquired TRAIL resistance we compared expression profiles of TRAIL-sensitive HL-60 cells and TRAIL-resistant HL-60 subclones using microarray mRNA gene expression chips and 2D-PAGE followed by MS and MS/MS analysis. Using cluster analysis of microarray mRNA gene expression profiles of 6 individual HL60 resistant subclones we identified two molecular signatures of TRAIL resistance. When compared to WT HL60 TRAIL-sensitive mRNA expression the group of HL60 subclones designed as phenotype 1 (P1, n=3) had 1122 significantly upregulated and 985 significantly downregulated mRNAs and subclones designed as phenotype 2 (P2, n=3) had 1583 significantly upregulated and 1798 significantly downregulated mRNAs (p<0.05). The subclones of P1 and P2 phenotype shared 533 upregulated and 422 downregulated genes compared to HL60 WT cells. To identify differentially expressed proteins we performed 2D-PAGE of two representative subclones of P1 and P2 phenotypes (in five replicates) and compared them with HL60 WT cells. We detected and identified differentially expressed proteins involved in various aspects of cellular metabolism. Except for downregulation of chromobox protein homolog 5, a protein involved in the regulation of gene expression, and downregulation of Annexin A6 and protein disulfide-isomerase A3 precursor the TRAIL-resistant P1 and P2 subclones showed different protein expression profile. In P1 TRAIL-resistant cells we identified downregulation of proteins involved in energy metabolism, including pyruvate kinase, enolase, cytochrome c-reductase, NADH dehydrogenase and downregulation of proteins involved in proteasome activity, such as proteasome activator complex subunits 1 and 2. In P2 TRAIL-resistant cells we detected downregulation of important regulatory proteins, such as DNA replication licensing factor MCM7, proliferation-associated protein 2G4, replication protein A 32 kDa subunit. In both, P1 and P2, subclones we identified significant changes in cytoskeletal rearrangement proteins. In addition, several chaperones were differentially expressed, namely protein-disulfide isomerase, GrpE, HSC71 and HSP27, 78 kDa glucose-regulated protein precursor. Our data show two HL60 cell line-derived TRAIL resistant subclones, P1 and P2, have specific molecular signatures suggesting distinct mechanisms of TRAIL resistance. These distinct pathways for development of resistance to TRAIL-induced apoptosis are relevant for design of more effective strategies for leukemia therapy.

Supported by MZCR 023736, MSM LC06044 and 0021620806, and IGA MZ NR8317 and NR8930.

Author notes

Disclosure: No relevant conflicts of interest to declare.