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Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

Morita R, Nishizawa S, Torigoe T, Takahashi A, Tamura Y, Tsukahara T, Kanaseki T, Sokolovskaya A, Kochin V, Kondo T, Hashino S, Asaka M, Hara I, Hirohashi Y, Sato N - Cancer Sci. (2014)

Bottom Line: In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC.Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC.A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.

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DNAJB8 gene overexpression experiments in HT29 cells. (a) Quantitative RT-PCR of DNAJB8 and colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) markers. Data are shown as a comparison with the expression level in HT29 control cells. Data represent means ± SD. (b) Isolation of side population (SP) cells. HT29 DNAJB8-overexpressed cells and control cells were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cell sorter. (c) Tumor growth of HT29 DNAJB8-overexpressed cells and control cells. HT29 DNAJB8-overexpressed cells, 103, and control cells were inoculated subcutaneously into the backs of five non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and tumor growth was measured weekly. Data represent means ± SD. The difference between HT29 DNAJB8-overexpression cells and control cells was examined for statistical significance using the Student's t-test. (d) Sphere formation assay. To assay sphere formation efficiency, 103 cells were cultured in Ultra Low Attachment six-well plates for 2 weeks and the number of spheres was counted under a microscope in 15 low-power fields and then the average was calculated.
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fig02: DNAJB8 gene overexpression experiments in HT29 cells. (a) Quantitative RT-PCR of DNAJB8 and colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) markers. Data are shown as a comparison with the expression level in HT29 control cells. Data represent means ± SD. (b) Isolation of side population (SP) cells. HT29 DNAJB8-overexpressed cells and control cells were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cell sorter. (c) Tumor growth of HT29 DNAJB8-overexpressed cells and control cells. HT29 DNAJB8-overexpressed cells, 103, and control cells were inoculated subcutaneously into the backs of five non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and tumor growth was measured weekly. Data represent means ± SD. The difference between HT29 DNAJB8-overexpression cells and control cells was examined for statistical significance using the Student's t-test. (d) Sphere formation assay. To assay sphere formation efficiency, 103 cells were cultured in Ultra Low Attachment six-well plates for 2 weeks and the number of spheres was counted under a microscope in 15 low-power fields and then the average was calculated.

Mentions: To evaluate the functions of DNAJB8 in HT29 cells, we established DNAJB8-overexpressed cells. We confirmed the overexpression of DNAJB8 mRNA using quantitative RT-PCR (Fig. 2a). Expression levels of the stem cell markers SOX2, LGR5 and POU5F1 were increased by 2.2-fold, 2.3-fold and 2.1-fold, respectively, in DNAJB8-overexpressed HT29 cells compared with the level in the control HT29 cells (Fig. 2a). The percentage of SP cells in DNAJB8-overexpressed HT29 cells was higher than that in control HT29 cells (Fig. 2b). To confirm the tumorigenicity in vivo of DNAJB8-overexpressed HT29 cells, we used a xenograft transplantation model. The DNAJB8-overexpressed HT29 cells showed higher tumor-initiating ability compared with HT29 control cells (Fig. 2c). A sphere formation assay was used to evaluate CSC/CIC-like features. DNAJB8-overexpressed HT29 cells showed higher sphere-forming ability than that of HT29 control cells (Fig. 2d), indicating that overexpression of DNAJB8 induced CSC/CIC properties.


Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

Morita R, Nishizawa S, Torigoe T, Takahashi A, Tamura Y, Tsukahara T, Kanaseki T, Sokolovskaya A, Kochin V, Kondo T, Hashino S, Asaka M, Hara I, Hirohashi Y, Sato N - Cancer Sci. (2014)

DNAJB8 gene overexpression experiments in HT29 cells. (a) Quantitative RT-PCR of DNAJB8 and colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) markers. Data are shown as a comparison with the expression level in HT29 control cells. Data represent means ± SD. (b) Isolation of side population (SP) cells. HT29 DNAJB8-overexpressed cells and control cells were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cell sorter. (c) Tumor growth of HT29 DNAJB8-overexpressed cells and control cells. HT29 DNAJB8-overexpressed cells, 103, and control cells were inoculated subcutaneously into the backs of five non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and tumor growth was measured weekly. Data represent means ± SD. The difference between HT29 DNAJB8-overexpression cells and control cells was examined for statistical significance using the Student's t-test. (d) Sphere formation assay. To assay sphere formation efficiency, 103 cells were cultured in Ultra Low Attachment six-well plates for 2 weeks and the number of spheres was counted under a microscope in 15 low-power fields and then the average was calculated.
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fig02: DNAJB8 gene overexpression experiments in HT29 cells. (a) Quantitative RT-PCR of DNAJB8 and colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) markers. Data are shown as a comparison with the expression level in HT29 control cells. Data represent means ± SD. (b) Isolation of side population (SP) cells. HT29 DNAJB8-overexpressed cells and control cells were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cell sorter. (c) Tumor growth of HT29 DNAJB8-overexpressed cells and control cells. HT29 DNAJB8-overexpressed cells, 103, and control cells were inoculated subcutaneously into the backs of five non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and tumor growth was measured weekly. Data represent means ± SD. The difference between HT29 DNAJB8-overexpression cells and control cells was examined for statistical significance using the Student's t-test. (d) Sphere formation assay. To assay sphere formation efficiency, 103 cells were cultured in Ultra Low Attachment six-well plates for 2 weeks and the number of spheres was counted under a microscope in 15 low-power fields and then the average was calculated.
Mentions: To evaluate the functions of DNAJB8 in HT29 cells, we established DNAJB8-overexpressed cells. We confirmed the overexpression of DNAJB8 mRNA using quantitative RT-PCR (Fig. 2a). Expression levels of the stem cell markers SOX2, LGR5 and POU5F1 were increased by 2.2-fold, 2.3-fold and 2.1-fold, respectively, in DNAJB8-overexpressed HT29 cells compared with the level in the control HT29 cells (Fig. 2a). The percentage of SP cells in DNAJB8-overexpressed HT29 cells was higher than that in control HT29 cells (Fig. 2b). To confirm the tumorigenicity in vivo of DNAJB8-overexpressed HT29 cells, we used a xenograft transplantation model. The DNAJB8-overexpressed HT29 cells showed higher tumor-initiating ability compared with HT29 control cells (Fig. 2c). A sphere formation assay was used to evaluate CSC/CIC-like features. DNAJB8-overexpressed HT29 cells showed higher sphere-forming ability than that of HT29 control cells (Fig. 2d), indicating that overexpression of DNAJB8 induced CSC/CIC properties.

Bottom Line: In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC.Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC.A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Show MeSH
Related in: MedlinePlus