Masamichi Ita

Comparative genomic hybridization reveals genetic progression of oral squamous cell carcinoma from dysplasia via two different tumourigenic pathways

To clarify the genetic pathway(s) involved in the development and progression of oral squamous cell carcinoma (OSCC), as well as the relationship between genetic aberrations and biological characteristics of OSCC tumours, comparative genomic hybridization was used to analyse genetic alterations in both primary OSCCs and adjacent dysplastic lesions of the same biopsy specimens from 35 patients. Gain of 8q22–23 was the most frequent alteration in both OSCC and mild dysplasia, and was considered the earliest event in the process of oral tumourigenesis. The average number of DNA sequence copy number aberrations (DSCNAs) increased with progression from mild dysplasia to invasive carcinoma (r = 0.737, n = 70, p < 0.001). OSCC samples were classified as having a large or small number of DSCNAs (OSCC‐L, 21.4 ± 4.7 DSCNAs or OSCC‐S, 10.0 ± 1.7 DSCNAs, respectively; p < 0.0001). Gains of 3q26‐qter, 8q, 11q13, 14q, and 20q and losses of 4q, 5q12–22, 6q, 8p, 13q, and 18q22‐qter were common to OSCC‐L and OSCC‐S. Gains of 5p15, 7p, 17q11–22, and 18p and losses of 3p14–21, 4p, and 9p were detected exclusively in OSCC‐L. The average number of DSCNAs depended on whether the samples showed OSCC‐ L or dysplasia plus OSCC‐L, or showed OSCC‐S or dysplasia plus OSCC‐S (p = 0.001). Gain of 5p15 and losses of 4p and 9p were detected even in dysplastic lesions adjacent to OSCC‐L samples. Loss of 4p was associated with node metastasis by multivariate analysis (p = 0.013). OSCC‐L tumours were more often T3–T4 stage tumours than T1–T2 stage tumours (p = 0.03). These findings suggest that two different types of OSCC, OSCC‐L associated with high‐stage cancer and OSCC‐S associated with low‐stage cancer, arise from different types of dysplasia via different genetic pathways. Copyright

Remarkable enhancement of cytotoxicity of onconase and cepharanthine when used in combination on various tumor cell lines

Onconase (Onc), a ribonuclease from oocytes or early embryos of Northern Leopard frog (Rana pipiens), is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and is currently in Phase IIIb clinical trials for malignant mesothelioma where it displays antitumor activity with minor overall toxicity to the patient. One of the characteristic features of Onc is a synergism with a variety of other antitumor modalities. Cepharanthine (Cep), a biscoclaurine alkaloid from Stephania cepharantha Hayata, is widely used in Japan to treat variety of ailments. It also shows low toxicity to patients. The aim of the present study was to assess the interaction of these two drugs on different tumor cell lines. When human promyelocytic leukemia HL-60, histiomonocytic lymphoma U937, multiple myeloma RPMI-8228, prostate carcinoma DU 145 and prostate adenocarcinoma LNCaP cells were exposed to relatively low concentrations of Onc or Cep their growth rates were somewhat suppressed but the cells were still able to proliferate. Cell growth, however, was totally abolished in each of these cell lines when treated with Onc and Cep combined. The frequency of apoptosis was also many-fold higher in cultures treated with a combination of Onc and Cep than in respective cultures treated with Onc or Cep alone. The mechanism of the observed synergism is unclear but it may be associated with the Onc activity in targeting microRNAs and/or NFkappaB and Cep activity also targeting NFkappaB. The data suggest that the combination of these two drugs, that individually express a low toxic profile, may have strong antitumor potential.

Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes.

BACKGROUND Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. MATERIALS AND METHODS Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G(0) to G(1) transition and the cell cycle progression. RESULTS During the first 24 h of stimulation, when the cells underwent G(0) to G(1) transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24-48 h of stimulation, when cells were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G(0) cells. CONCLUSIONS While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G(0) to G(1) transition, the cells progressing through the remainder of the cycle are better distinguished from G(0) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments.

KRN5500, a novel antitumor agent, induces apoptosis or cell differentiation in HL‐60 cells

BACKGROUND KRN5500, a derivative of spicamycin, shows antitumor activity against a variety of tumor cell lines. However, the mechanism of cytotoxic action has remained unclear. METHODS The viability of HL-60 human leukemic cells treated with KRN5500 was studied by the dye exclusion assay. Induction of apoptosis and effects on the cell cycle were investigated by flow cytometry: We measured cellular DNA content after extraction of fragmented DNA, and apoptosis-induced DNA strand breaks. Cell morphology was observed by light microscopy. DNA strand breaks at a nucleosomal unit were analyzed by electrophoresis. RESULTS Our data demonstrated that KRN5500 caused inhibition of cell growth, and that apoptosis was the mode of cell death. G(1) phase cells were more susceptible to KRN5500 induced apoptosis. In addition, KRN5500 induced cell differentiation at lower concentration. CONCLUSIONS It is anticipated that KRN5500 will be used clinically as an anti-leukemic agent. Its mechanism of antitumor action is to induce apoptosis or cell differentiation.

A signaling pathway by a new synthetic lipid A analog, ONO-4007, in RAW264.7 cells

ONO-4007 is a new synthetic lipid A derivative with low endotoxic activities. We have reported that ONO-4007 could be a new bio-logical response modifier for the treatment of tumor necrosis factor (TNF)-&agr;-sensitive tumors. In this study, we confirmed that ONO-4007 activated a murine macrophage cell line, RAW264.7, and that the activated RAW264.7 cells produced TNF-&agr; in vitro. RAW264.7 cells stimulated for less than 15 min with ONO-4007 (40 μg/ml) did not produce TNF-&agr; (less than 4 U/ml). However, 24 h stimulation with ONO-4007 induced TNF-&agr; production (more than 256 U/ml) in RAW264.7 cells. Although P38 in mitogen-activated protein kinase of the RAW264.7 cells was not tyrosine phosphorylated by ONO-4007 stimulation, ERK1 of the RAW264.7 cells was tyrosine phosphorylated for 5–15 min by ONO-4007 stimulation. Tyrosine phosphorylation of ERK1 decreased gradually from 15 min after stimulation and almost disappeared 60 min after stimulation. These findings indicate that ONO-4007 stimulates RAW264.7 cells immediately and induces tyrosine phosphorylation of ERK1 in the RAW264.7 cells for 5–15 min. These data suggest that the signal transduction pathway of ONO-4007 may be similar to that of lipopolysaccharide.