[Clinical phase I trial of S-1 in the combination with DOC using super-selective intra-arterial infusion with oral cancer].

The super-selective intra-arterial infusion, which has high anti-tumor effect to infuse high concentration of drugs into arterial in the control of tumor, has been expected to have local control. S-1, developed by the scientific theory of both potentiating antitumor activity of 5-fluorouracil(5-FU)and reducing gastrointestinal toxicity induced by 5-FU, is an active agent for squamous cell carcinoma of the head and neck(HNSCC). Docetaxel(DOC)is the drug Taxanes which has anti-tumor effect by mechanism different from conventional anti-tumor mechanism of action. In Yamaguchi University, DOC+CDDP+5-FU in the three-drug combination therapy shows high anti-tumor effect for advanced oral cancer. In the present study, we conducted a phase I study to examine local control of S-1 in the combination with DOC using super-selective intra-arterial infusion with oral cancer. The study performed super-selective intra-arterial infusion of DOC on the first day, and was considered as the schedule which prescribes three-week S-1 for patients every day from same day. Since blood toxicity nature grade 4 discovered the result in S-1: 65 mg/m(2)day and DOC: 50 mg/ m(2), we decided that recommended dose(RD)was S-1: 65 mg/m(2) and DOC: 40 mg/m(2).

Molecular cytogenetic analysis of oral squamous cell carcinomas by comparative genomic hybridization, spectral karyotyping, and fluorescence in situ hybridization.

We investigated relationships between DNA copy number aberrations and chromosomal structural rearrangements in 11 different cell lines derived from oral squamous cell carcinoma (OSCC) by comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). CGH frequently showed recurrent chromosomal gains of 5p, 20q12, 8q23 approximately qter, 20p11 approximately p12, 7p15, 11p13 approximately p14, and 14q21, as well as losses of 4q, 18q, 4p11 approximately p15, 19p13, 8p21 approximately pter, and 16p11 approximately p12. SKY identified the following recurrent chromosomal abnormalities: i(5)(p10), i(5)(q10), i(8)(q10), der(X;1)(q10;p10), der(3;5)(p10;p10), and der(3;18)(q10;p10). In addition, breakpoints detected by SKY were clustered in 11q13 and around centromeric regions, including 5p10/q10, 3p10/q10, 8p10/q10 14q10, 1p10/1q10, and 16p10/16q10. Cell lines with i(5)(p10) and i(8)(q10) showed gains of the entire chromosome arms of 5p and 8q by CGH. Moreover, breakages near the centromeres of chromosomes 5 and 8 may be associated with 5p gain, 8q gain, and 8p loss in OSCC. FISH with a DNA probe from a BAC clone mapping to 5p15 showed a significant correlation between the average numbers of i(5)(p10) and 5p15 (R(2) = 0.8693, P< 0.01) in these cell lines, indicating that DNA copy number of 5p depends upon isochromosome formation in OSCC.

Detection of DNA strand breaks by flow and laser scanning cytometry in studies of apoptosis and cell proliferation (DNA replication).

Extensive fragmentation of nuclear DNA occurs during apoptosis, and the presence of DNA strand breaks is considered to be a marker of the apoptotic mode of cell death. This chapter describes methods to label in situ DNA strand breaks with fluorochromes for detection by flow or laser scanning cytometry. By staining DNA with a fluorochrome of another color, cellular DNA content is measured concurrently and the bivariate analysis of such a data reveals DNA ploidy and cell-cycle phase position of apoptotic cells. The DNA strand break-labeling methodology is also used for detecting the incorporation of halogenated DNA precursors in studies of the cell cycle, proliferation, and DNA replication. In this application, termed "strand breaks induced by photolysis" (SBIP), the cells are incubated with 5-bromo-2'-deoxyuridine (BrdU) to incorporate it into DNA and sensitize the DNA to ultraviolet (UV) light. DNA strand breaks are then photolytically generated by exposing the cells to UV light. The DNA strand breaks resulting from UV-photolysis are subsequently fluorochrome-labeled as for labeling apoptotic-DNA breaks. Because SBIP, unlike the alternative method of detection of BrdU incorporation, does not require subjecting cells to harsh conditions (strong acid or heat) of DNA denaturation, it is compatible with concurrent detection of intracellular or cell surface antigens by immunocytochemical means.

Histone H2AX phosphorylation induced by selective photolysis of BrdU-labeled DNA with UV light: relation to cell cycle phase.

BACKGROUND: The induction of DNA double-strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser-139) by ATM-, ATR-, or DNA-dependent protein kinases (DNA-PK). Phosphorylated H2AX, denoted as gammaH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser-139-phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring gammaH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of gammaH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. METHODS: HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU-labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU-labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for gammaH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6-diamidino-2-phenylindole. The intensities of cellular far red (gammaH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. RESULTS: After a 1-h pulse of BrdU followed by exposure to UV, nearly all cells with S-phase DNA content had many-fold higher gammaH2AX IF than G(1) or G(2)/M cells. The nonirradiated cells had minimal ("programmed") expression of gammaH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of gammaH2AX IF independent of the cell cycle phase. Pulse-chase experiments showed that the cohort of BrdU-labeled (gammaH2AX-positive) cells progressed through G(2)/M and into G(1) phase after 8 and 12 h of growth in BrdU-free medium, respectively. Bivariate analysis of gammaH2AX versus cyclin A expression for the gated S-phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. CONCLUSIONS: Photolysis of BrdU-labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of gammaH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics.

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

BACKGROUND: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. RESULTS: In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS: Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Concurrent chemoradiotherapy with new platinum compound nedaplatin in oral cancer.

Eleven patients with oral cancer were treated with the new platinum agent, nedaplatin, and 5-fluorouracil and simultaneous radiation therapy. The regimen was of 5 days' duration: 5-fluorouracil 400-500 mg/m(2) (days 1-5), nedaplatin 80-90 mg/m(2) (day 4), and a total radiation dose of 8-10 Gy (days 1-5). Of the 11 patients, 4 (36.4%) showed complete response and 5 (45.4%) showed partial response; the total response rate was 81.8%. Major side effects were hypochromia (27%), leukopenia (64%), granulocytopenia (55%), thrombocytopenia (36%), nausea and vomiting (82%), and mucositis (45%). Toxicity was grade 2 or less in most of the 11 patients. The results indicate that the regimen composed of combination chemotherapy with nedaplatin and radiation therapy is effective in the treatment of patients with squamous cell carcinoma of the oral region.