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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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Isolation of colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) as side population (SP) cells. (a) Isolation of SP cells from colon cancer cell lines. SW480, HCT15 and HT29 were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cells sorter. (b) RT-PCR of DNAJB8. MP, main population. (c) RT-PCR of CSC/CIC markers in HT29 cells. (d) Tumor growth of HT29 SP cells and MP cells. HT29 SP cells, 103, and MP 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 SW480 SP and MP cells was examined for statistical significance using the Student's t-test.
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fig01: Isolation of colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) as side population (SP) cells. (a) Isolation of SP cells from colon cancer cell lines. SW480, HCT15 and HT29 were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cells sorter. (b) RT-PCR of DNAJB8. MP, main population. (c) RT-PCR of CSC/CIC markers in HT29 cells. (d) Tumor growth of HT29 SP cells and MP cells. HT29 SP cells, 103, and MP 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 SW480 SP and MP cells was examined for statistical significance using the Student's t-test.

Mentions: Human CRC CSC/CIC have previously been isolated from CRC cell lines and primary CRC samples using several methods, including the use of cell surface markers (e.g. CD133), SP cells using Hoechst 33342 analysis and the Aldefluor assay.(22–26) In the present study, we isolated CRC CSC/CIC using SP cell analysis. Although previous studies showed that some colon cancer SP cells were not enriched with a CSC/CIC population,(27) our previous study confirmed that SW480, HT29 and HCT15 SP cells had high tumor-initiating ability, high expression levels of stem cell markers such as SOX2 and POU5F1 and strong resistance to chemotherapeutic agents compared with MP cells.(18) The ratio of SW480 SP cells was 4.0%, that of HCT15 cells was 5.5% and that of HT29 cells was 0.8%. All of these SP cells were specifically inhibited by verapamil, an ABC transporter inhibitor (Fig. 1a). Expression of DNAJB8 mRNA in SP cells and MP cells was examined using RT-PCR. DNAJB8 mRNA was detected in SP cells derived from SW480, HCT15 and HT29 cells, whereas DNAJB8 mRNA was not detected in MP cells (Fig. 1b). Thus, DNAJB8 is one candidate of CSC/CIC-specific antigens in CRC as well as in renal cell carcinomas. DNAJB8 mRNA was detected in SP cells derived from HT29 cells at the highest level (Fig. 1b) and therefore HT29 cells were used for further analysis. The quality of SP cells derived from HT29 cells as a source of CRC CSC/CIC was confirmed by higher expression levels of stem cell markers, including SOX2, POU5F1 and LGR5, using RT-PCR and by a higher tumor-initiating ability in a xenograft model than those of MP cells (Fig. 1c, d).


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)

Isolation of colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) as side population (SP) cells. (a) Isolation of SP cells from colon cancer cell lines. SW480, HCT15 and HT29 were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cells sorter. (b) RT-PCR of DNAJB8. MP, main population. (c) RT-PCR of CSC/CIC markers in HT29 cells. (d) Tumor growth of HT29 SP cells and MP cells. HT29 SP cells, 103, and MP 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 SW480 SP and MP cells was examined for statistical significance using the Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317808&req=5

fig01: Isolation of colon cancer stem-like cells/cancer-initiating cells (CSC/CIC) as side population (SP) cells. (a) Isolation of SP cells from colon cancer cell lines. SW480, HCT15 and HT29 were stained with Hoechst 33342 dye with or without verapamil and analyzed using a FACSAria II cells sorter. (b) RT-PCR of DNAJB8. MP, main population. (c) RT-PCR of CSC/CIC markers in HT29 cells. (d) Tumor growth of HT29 SP cells and MP cells. HT29 SP cells, 103, and MP 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 SW480 SP and MP cells was examined for statistical significance using the Student's t-test.
Mentions: Human CRC CSC/CIC have previously been isolated from CRC cell lines and primary CRC samples using several methods, including the use of cell surface markers (e.g. CD133), SP cells using Hoechst 33342 analysis and the Aldefluor assay.(22–26) In the present study, we isolated CRC CSC/CIC using SP cell analysis. Although previous studies showed that some colon cancer SP cells were not enriched with a CSC/CIC population,(27) our previous study confirmed that SW480, HT29 and HCT15 SP cells had high tumor-initiating ability, high expression levels of stem cell markers such as SOX2 and POU5F1 and strong resistance to chemotherapeutic agents compared with MP cells.(18) The ratio of SW480 SP cells was 4.0%, that of HCT15 cells was 5.5% and that of HT29 cells was 0.8%. All of these SP cells were specifically inhibited by verapamil, an ABC transporter inhibitor (Fig. 1a). Expression of DNAJB8 mRNA in SP cells and MP cells was examined using RT-PCR. DNAJB8 mRNA was detected in SP cells derived from SW480, HCT15 and HT29 cells, whereas DNAJB8 mRNA was not detected in MP cells (Fig. 1b). Thus, DNAJB8 is one candidate of CSC/CIC-specific antigens in CRC as well as in renal cell carcinomas. DNAJB8 mRNA was detected in SP cells derived from HT29 cells at the highest level (Fig. 1b) and therefore HT29 cells were used for further analysis. The quality of SP cells derived from HT29 cells as a source of CRC CSC/CIC was confirmed by higher expression levels of stem cell markers, including SOX2, POU5F1 and LGR5, using RT-PCR and by a higher tumor-initiating ability in a xenograft model than those of MP cells (Fig. 1c, d).

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