Therapeutic Agent for Neuroblastoma Targeting ARID3b
a neuroblastoma and therapy agent technology, applied in the field of compositions for treating neuroblastoma, can solve the problems of preventing the development of a new treatment method, affecting the treatment effect of patients of one year or older, and affecting the treatment effect of patients
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example 1
[0047]Expression of ARID3b mRNA was examined for five neuroblastoma cell lines. SH-SY5Y (ATCC CRL-2266), TGW (Iwasaki, I. et al., Cancer Chemother. Pharmacol., 49:438-444 (2002)), CHP-126 (Schlesinger, H. R. et al., Cancer Res., 36:3094-3100 (1976)), NBLS and IMR (ATCC CCL-127) were used as neuroblastoma cell lines. Furthermore, K562 (chronic myelogenous leukemia, ATCC CCL-243) was used as a control. A medium consisting of 45% Dulbecco's minimum essential medium (D-MEM, Invitrogen), 45% Ham's F-12 medium (Invitrogen) and 10% fetal bovine serum (FBS, JRL) was used for the cultivation of neuroblastoma cell lines. A medium consisting of 90% D-MEM and 10% FBS was used for other cell lines. RNA was extracted from each cell line according to a conventional method, and subjected to Northern blot hybridization using human ARID3b DNA (SEQ ID NO:8) as a probe. Detection of actin mRNA was carried out in a similar manner. The results are shown in FIG. 1A. As shown in FIG. 1A, although the expre...
example 2
[0050]Influence of forced expression of ARID3b on in vitro malignancy of a cell was examined using growth ability and colony formation ability as indexes. A retrovirus having human ARID3b DNA (SEQ ID NO:8) being inserted was constructed. The retrovirus contained in MSCV Retrovirus Expression System (Cat. No. 634401) which is commercially available from Clontech was used as a retrovirus after substituting IRES GFP for the drug resistance gene portion. This recombinant retrovirus was used to infect the neuroblastoma cell line SH-SY5Y to forcibly express ARID3b and the cell number was measured over time. For SH-SY5Y, expression of ARID3b was observed in Example 1, and it was known that amplification of MYCN oncogene is not found. The results are shown in FIG. 2A. In the figure, diamonds (♦) represent results of infection with the retrovirus having ARID3b DNA being inserted, and squares (▪) represent results of infection with the vector without the insert DNA. As shown in FIG. 2A, the c...
example 3
[0054]Influence of forced expression of ARID3b on canceration of a primary cultured cell was examined using senescence and immortalization during passage cultures as indexes. The retrovirus having human ARID3b DNA being inserted used in Example 2 was used to infect mouse primary cultured fibroblasts isolated from a fetus. A medium consisting of 90% D-MEM and 10% FBS was used for the cultivation of primary cultured fibroblasts. The cells were passaged at intervals of two or three days by washing with Dulbecco's phosphate-buffered saline (PBS, Invitrogen), detaching from dishes using 0.05% (w / v) trypsin-EDTA (Invitrogen), and diluting 5-fold. The cells were counted upon passages. The results are shown in FIG. 4. In the figure, diamonds (♦) represent results of infection with the retrovirus having ARID3b DNA being inserted and squares (▪) represent results of infection with the vector without the insert DNA. As shown in FIG. 4, when the primary cultured fibroblasts infected with the ve...
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