Kazuhiro Yoshitani

Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines

Loss of p16INK4a protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1α for p16INK4a involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16INK4a. In this study, we focused on the expression and methylation status in the regions of nt −478 to −201, containing a putative TATA box (nt −401 to −396), and nt −233 to 26, both in a recently cloned 5′ upstream region of rat p16INK4a. We showed that rat lung adenocarcinoma RLCNR did not express the p16INK4a gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt −233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5‐aza 2′‐deoxycytidine induced expression of p16INK4a gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16INK4a gene, rather than that of other regions.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation.

Allogeneic and autologous stem cell transplantation in advanced small round cell sarcomas

Small round cell sarcomas (SRSs) comprise a heterogeneous group of malignant neoplasms with similar cytomorphology characterized by small, round, relatively undifferentiated cells; the group includes Ewing family sarcomas and rhabdomyosarcoma. These malignancies are chemotherapy-sensitive and potentially curable, and are treated by multimodality, dose-intensive, neoadjuvant protocols. For Ewing family sarcomas and rhabdomyosarcoma, actinomycin D-based chemotherapy combined with vincristine and cyclophosphamide is widely accepted as a standard therapy regimen. Since the beginning of the 1970s, such regimens have signifi cantly improved the patients’ prognosis. However, even after induction of these regimens, few patients with overt metastasis at diagnosis or with localized but extensive unresectable primary lesions of the trunk are cured. Most high-risk patients initially respond to treatment, but most of them eventually suffer a recurrence of the tumor and die of disseminated disease. To try to improve systemic control, hematopoietic stem cell transplantation (HSCT) has been considered as a possible approach. Autologous blood stem cell transplantation, such as peripheral blood cell transplantation (PBSCT), rescues patients who are myelosuppressed following high-dose chemotherapy. Allogeneic blood stem cell transplantation, such as umbilical cord blood transplantation (UCBT), may have the advantage of having graft-versus-tumor (GVT) effects. Here, we report two cases of patients with SRS who suffered a refractory recurrence and systemic metastasis of their tumor. Without any consolidation therapy, we could not prevent complete paralysis (case 1) or general dissemination (case 2). We performed high-dose chemotherapy rescued by autologous PBSCT (auto-PBSCT) and UCBT. Our treatment produced a marked response and induced a partial remission. We describe the courses of the patients and the residual problems associated with our treatment. All the treatments were performed after informed consent was obtained from the patients and their families, and approval of the chief of our institution was obtained according to the institutional code of ethics. The patients and their families were informed that data from the case would be submitted for publication and gave their consent.